Methods for inhibiting endothelial cell and fibrinogen mediated inflammation using fibrinogen specific antibodies

ABSTRACT

The present invention contemplates therapeutic compositions containing a fibrinogen homolog capable of binding to endothelial cells in an RGD-independent manner that inhibits fibrinogen binding to endothelial cells. Also described are therapeutic compositions containing an ICAM-1 homolog capable of binding to fibrinogen in an RGD-independent manner that inhibits fibrinogen binding to endothelial cells. Methods of inhibiting endothelial cell and fibrinogen mediated inflammation within a patient by administering a homolog of this invention are also contemplated.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. Patent application Ser. No.09/347,877 filed Jul. 6, 1999, now issued as U.S. Pat. No. 6,265,549,which is a divisional of U.S. patent application Ser. No. 08/748,150filed Nov. 12, 1996, now issued as U.S. Pat. No. 5,919,754, which is adivisional of Ser. No. 08/232,532 filed Apr. 25, 1994, now issued asU.S. Pat. No. 5,599,790, which is a continuation-in-part of U.S. patentapplication Ser. No. 08/139,562 filed Oct. 19, 1993 now abandoned whichis a continuation of U.S. patent application Ser. No 07/898,117 filedJun. 2, 1992, now abandoned.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant Nos. HL43773 and HL 51372 awarded by the National Institutes of Health.

TECHNICAL FIELD

The present invention contemplates the use of compositions to inhibitfibrinogen binding to endothelial cells for the purpose of inhibitingendothelial cell and fibrinogen mediated inflammation.

BACKGROUND

Adhesion of leukocytes to vascular endothelium is one of the earliestevents in a variety of immune-inflammatory reactions. The processparticipates in vascular occlusions and contributes to atherothromboticlesions. At the molecular level, leukocyte adhesion to endothelial cellsis a redundant mechanism, supported by the regulated recognition of adisparate set of membrane receptors, including integrins, expressed onboth leukocytes and resting or cytokine-activated endothelial cells.

Integrins are a functionally and structurally related group of receptorsthat interact with a wide variety of ligands including extracellularmatrix glycoproteins, complement and other cells. Integrins participatein cell-matrix and cell-cell adhesion in many physiologically importantprocesses including embryological development, hemostasis, thrombosis,wound healing immune and nonimmune defense mechanisms and oncogenictransformation. See Hynes, Cell, 48:549-554 (1987). The majority ofintegrins participating in dynamic cell adhesion, bind a tripeptide,arginine-glycine-aspartic acid (RGD), present in their ligand, causingcell adhesion. See Ruoslahti et al., Science, 238:491-497 (1987).

Mac-1 (CD11b/CD18) is an integrin receptor found predominantly onmacrophages and granulocytes. Like all integrin receptors, Mac-1 is aheterodimeric, transmembrane glycoprotein composed of non-covalentlyassociated alpha and beta subunits.

Mac-1 mediates neutrophil/monocyte adhesion to vascular endothelium andphagocytosis of complement-opsonized particles. Antibodies to the Mac-1receptor alter neutrophil function in vivo including inhibitingneutrophil migration into inflammatory sites. See Price et al., J.Immunol., 139:4174-4177 (1987). Mac-1 also functions as a receptor forfibrinogen in a reaction linked to fibrin deposition on the monocytesurface. See Altieri et al., J. Cell Biol., 107:1893-1900 (1988); Wrightet al., Proc. Natl. Acad. Sci. USA, 85:7734-7738 (1988); Trezzini etal., Biochem. Biophys. Res. Commun., 156:477-484 (1988) and Gustafson etal., J. Cell Biol., 109:377-387 (1989).

Fibrinogen is a complex molecule of approximately 340,000 daltons andconsists of three pairs of subunit polypeptides, called the α, β and γchains. These individual chains are held together by several disulfidebonds. The proteolytic digestion of fibrinogen by plasmin producesfragments A, B, C, D and E, all having a molecular weight of less the85,000 daltons. See Pizzo et al., J. Biol. Chem., 247:636-645 (1972).

Further proteolytic digestion of fibrinogen by plasmin produces a D₃₀fragment with a molecular weight of about 30,000 daltons containingportions of the α, β and γ chains of fibrinogen. See Furlan et al.,Biochim. Biophys. Acta., 400:95-111 (1975).

The deposition of fibrinogen on the leukocyte surface occurs in avariety of inflammatory responses such as delayed type hypersensitivity,incompatible transplant rejection and the physiopathology of vascularobstruction and atherogenesis. See Geczy et al., J. Immunol.,130:2743-2749 (1983); Hooper et al., J. Immunol., 126:1052-1058 (1981);Colvin et al., J. Immunol., 114:377-387 (1975); Hattler et al., CellImmunology, 9:289-295 (1973); Gerrity, R. G., Am. J. Pathol.,103:181-190 (1981) and Am. J. Pathol., 103:191-200 (1981); and Shelleyet al., Nature, 270:343-344 (1977).

Interactions of fibrinogen on cell surface receptors of endothelialcells have been described. Languino et al., Blood, 73:734 (1989)describe the binding of fibrinogen to endothelial cells by anRGD-dependent mechanism. It is generally believed that the vitronectinreceptor is the major endothelial receptor for fibrinogen. Cheresh etal, Proc. Natl. Acad. Sci. USA, 84:6471-6475 (1987). Other endothelialcell receptors reported to bind fibrinogen include cell surface boundtransglutaminase, and an 130 kilodalton receptor that binds to fibrinpeptides. Erban et al., J. Biol. Chem., 267:2451 (1992).

Also on the surface of endothelial cells is an intercellular adhesionmolecule 1 (ICAM-1) that has been described by Springer, Nature,346:425-433 (1990), and has been shown to bind the leukocyte integrinLFA-1.

Recently, the interaction of fibrinogen with the Mac-1 receptor ofleukocytes has been shown to be a dynamic cell adhesion reactioninvolving the recognition of the tripeptide RGD within fibrinogen by theMac-1 receptor similar to the interaction of fibrinogen with theintegrin receptors on platelets and endothelial cells. See Altieri etal., J. Clinic Invest., 78:968-976 (1986); Pytela et al., Science,231:1559-1562 (1986); Ruoslahti et al., Science, 238:491-497 (1987);Ruoslahti et al., Cell, 44:517-518 (1986); and International PCTApplication No. PCT/US 91/05096.

BRIEF DESCRIPTION OF THE INVENTION

It has now been discovered that fibrinogen binds to both the Mac-1receptor on leukocytes and to an endothelial cell receptor (ECR),thereby bridging between the leukocyte and the endothelial cell duringthe process of inflammation. Inflammation arising from this bridgingevent is referred to as endothelial cell/fibrinogen-mediatedinflammation. The ECR is an RGD-independent, fibrinogen specificreceptor.

The invention describes novel compositions defining the binding sitesfor the interaction between ECR and fibrinogen.

Thus, a composition is contemplated comprising a therapeuticallyeffective amount of a substantially pure and pharmaceutically acceptablefibrinogen homolog capable of binding to ECR and inhibiting Fg bindingto endothelial cells. In preferred embodiments, ECR is ICAM-1.

A preferred Fg homolog is a polypeptide Fg homolog having an amino acidresidue sequence derived from fibrinogen. A preferred polypeptide has atotal sequence of from about 17 to 100 amino acid residues in lengththat includes the fibrinogen γ chain sequence from fibrinogen γ chainresidues 117-133, which γ chain sequence is shown in SEQ ID NO 2, andthe polypeptide is capable of binding to ICAM-1 and inhibitingfibrinogen binding to endothelial cells, variants thereof, andcompositions containing a Fg polypeptide homolog.

Also contemplated is an antibody that immunoreacts with a Fg polypeptidehomolog as described herein. The antibody also immunoreacts withfibrinogen and inhibits fibrinogen binding to endothelial cells.

Also contemplated is a composition comprising a therapeuticallyeffective amount of a substantially pure and pharmaceutically acceptableICAM-1 homolog capable of binding to fibrinogen and inhibitingfibrinogen binding to endothelial cells.

The invention also describes a monoclonal antibody that immunoreactswith ICAM-1, but does not immunoreact with the vitronectin receptor,such that the monoclonal antibody preferentially inhibits fibrinogenbinding to stimulated endothelial cells.

A monoclonal antibody is also described that immunoreacts withfibrinogen and that preferentially inhibits fibrinogen binding tostimulated endothelial cells.

Also described is a method of inhibiting fibrinogen (Fg) binding toendothelial cells comprising contacting the endothelial cells with aFg-binding inhibiting amount of a physiologically acceptable compositioncomprising a homolog selected from the group consisting of a Fg homologand an ICAM-1 homolog.

The method is useful for inhibiting fibrinogen/endothelial cell-mediatedinflammation in a patient and comprises administering to a patient atherapeutically effective amount of a pharmaceutically acceptablecomposition comprising a substantially pure homolog selected from thegroup consisting of a Fg homolog and an ICAM-1 homolog.

The invention also describes a method of detecting the amount of afibrinogen (Fg) homolog in a liquid sample comprising:

(a) admixing a sample of stimulated endothelial cells with apredetermined amount of a liquid sample containing a Fg homolog and apredetermined amount of labelled Fg homolog to form a competitionreaction admixture;

(b) maintaining the reaction admixture for a predetermined time periodsufficient for any Fg homolog present in said composition to bind to theendothelial cells and form an endothelial cell:Fg homolog complex and toallow the labelled Fg homolog to bind to the endothelial cells to form alabelled endothelial cell:Fg homolog complex; and

(c) assaying for the amount of labelled endothelial cell:Fg homologcomplex formed in step (b) thereby detecting the amount of a Fg homologin the composition.

Also described is a method of screening for compositions effective atinhibiting fibrinogen binding to ICAM-1 comprising the steps of:

a) admixing in an inhibition reaction admixture preselected amounts of aputative inhibitor composition, a fibrinogen homolog, and an ICAM-1homolog;

b) maintaining the admixture under conditions sufficient for the ICAM-1homolog to bind to the Fg homolog and form an ICAM-1 homolog:Fg homologcomplex; and

c) measuring the amount of ICAM-1 homolog:Fg homolog complex formed instep (b), and thereby the effectiveness of the inhibitor composition.

Also described is a method for preparing substantially pure ICAM-1comprising the steps of:

(a) providing an aqueous detergent composition containing at leastICAM-1;

(b) contacting the ICAM-1-containing composition with afibrinogen-immobilized matrix comprising fibrinogen affixed to a solidsupport, wherein the contacting is conducted under conditions sufficientfor the ICAM-1 to bind to the fibrinogen and form a solid phaseICAM-1:fibrinogen complex;

(c) washing the solid support and the complex with an aqueous washbuffer comprising Mg⁺⁺, Mn⁺⁺ and an RGD-containing polypeptide underconditions sufficient to elute any proteins bound to fibrinogen in anRGD-dependent manner, wherein the wash buffer is substantially free fromCa⁺⁺; and

(d) eluting the ICAM-1 from the solid support using an aqueous buffercomprising Mg⁺⁺, Mn⁺⁺ and EDTA, to form the substantially pure ICAM-1.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, forming a portion of this disclosure:

FIG. 1 illustrates the autoradiographic results of electrophoresis ofaliquots of peak fractions from both the RGD and EDTA elutions of celllysate supernatants prepared from cells either left untreated or treatedwith TNF as described in Example 2A. Lanes 1 and 3 show the RGD-elutedreceptors isolated from cell lysates respectively prepared fromuntreated or TNF-treated cells. The characteristic pattern of VNR ispresent in lane 3. Lanes 2 and 4 respectively show the EDTA-eluted ECRisolated from untreated and TNF-treated cells having a molecular weightband of approximately 90-95 kD, the intensity of which is enhanced about3-5 fold as a result of the induction of ECR expression by exposure toTNF.

FIG. 2 illustrates the results of autoradiographic exposure of theelectrophoresed ¹²⁵I-labelled receptors isolated from sequentialaffinity chromatography of ¹²⁵I-labelled HUVEC cell lysate supernatantsover an RGD Sepharose™ followed by a fibrinogen Sepharose™ column. Thelanes are labelled 1-8. Lanes 1 through 4 show migration of proteinsunder reducing conditions while lanes 5 through 8 show the migration ofidentical aliquots run under nonreducing conditions. ¹²⁵I-labelledmolecular weight standards of 210, 107, 71 and 41 kD, respectively,myosin, beta-galactosidase, bovine serum albumin and ovalbumin are runin lanes 4 and 8.

In lanes 3 and 7, the vitronectin receptor eluted with EDTA from theRGD-Sepharose™ column exhibits the characteristic profile of alphav/beta 3 under reducing and nonreducing conditions as described inExample 2C. Another integrin beta subunit, beta 1, also shown in lanes 3and 7, was eluted from the RGD Sepharose™ column with EDTA.

Lanes 1 and 5 show the RGD elution of the flow-through from the firstRGD column applied onto the second fibrinogen column. Lane 6 shows theresults of EDTA elution following the RGD elution where a single band ofapproximately 90-95 kD under nonreducing conditions was recovered. Underreducing conditions, the molecular weight of the EDTA-eluted fibrinogenreceptor only slightly increased as shown in lane 2.

FIG. 3 illustrates the dose-response curve of ¹²⁵I-labelled fibrinogenbinding to monolayers of HUVEC as described in Example 3A2). The¹²⁵I-labelled fibrinogen bound in counts per minute (cpm) per well (X10⁻³) is plotted on the Y-axis against increasing concentrations of¹²⁵I-labelled fibrinogen (X 10⁻⁷ M) on the X-axis. The data shows that¹²⁵I-labelled fibrinogen binds saturably at a concentration ofapproximately 0.36 μM to monolayers of unstimulated HUVEC.

FIG. 4 illustrates the dose-response curve of ¹²⁵I-labelled fibrinogenbinding to unstimulated and either TNF- or LPS-stimulated HUVEC asdescribed in Example 3A3). ¹²⁵I-labelled fibrinogen bound in moleculesper cell (X 10⁻⁶) is plotted on the Y-axis against increasingconcentrations of ¹²⁵I-labelled fibrinogen (X 10⁻⁷ M) on the X-axis.Under stimulation with either TNF or LPS, the number of labelledfibrinogen molecules bound per cell doubled in comparison to those boundto unstimulated cells.

FIGS. 5A and 5B illustrate the dose and time dependent effects on theability of fibrinogen to mediate the binding of ⁵¹Cr-labelled THP-1cells to HUVEC. The results of these experiments are shown in FIG. 5Aand FIG. 5B where the data is expressed as numbers of ⁵¹Cr-labelledTHP-1 cells (X 10⁻³) on the Y-axis plotted against the assay time on theX-axis. FIG. 5A shows the effect of different concentrations of purifiedfibrinogen admixed with THP-1 cells compared to the absence offibrinogen (labelled as Fg) in mediating the binding to HUVEC culturesover a 60 minute period as described in Example 3B1). FIG. 5B showsresults of similar assays done in the presence of dilutions of normalhuman plasma (NHP).

FIG. 6 illustrates the results of the Western blot as described inExample 4E. Radiolabelled molecular weight markers of 97, 66, 45, 30 and21 kD shown in lane left of the first set of 5 Daudi lanes and left ofthe second set of 8 HUVEC lanes. Lanes designated 1-5 at the bottom ofthe blot for both Daudi and HUVEC were respectively immunoreacted with2E1, PMI -I, affinity purified 14E11, 14E11 culture supernatants and theanti-ICAM-1 BD monoclonal antibodies. The three extra lanes in the HUVECside of the blot show the nonspecific background when no primaryantibody is used.

FIG. 7 illustrates in bar graphs the inhibition of binding of¹²⁵I-labelled fibrinogen to HUVEC cultures in the presence of 50 foldexcess of unlabelled fibrinogen (Fg) over time as described in Example5A1). The amount of radioactivity associated with the cells afterharvesting is expressed on the Y-axis as cpm/well (X 10⁻³). Thenoninhibitory effects of exposure to the monoclonal antibodies directedagainst VNR, designated mAb 609 and mAb 7E3, is also shown. Totalbinding of ¹²⁵I-labelled fibrinogen in the absence of admixed inhibitorsis also shown.

FIG. 8 illustrates the effects of exposure to RGD- and RGE-containingpeptides on the binding of 125I-labelled fibrinogen to HUVEC asdescribed in Example 5A1). The amount of ¹²⁵I-labelled fibrinogen boundto HUVEC in cpm/well (X 10⁻³) is plotted on the Y-axis against thelength of time labelled fibrinogen was maintained with HUVEC.

FIGS. 9A and 9B illustrate the inhibition of ¹²⁵I-labelled fibrinogen tounstimulated or TNF-stimulated HUVEC cultures, respectively, FIGS. 9Aand 9B, by treatment of the HUVEC with the monoclonal antibodies, 14E11,BD (anti-ICAM-1, Becton Dickinson) and a control antibody, PMI-I. Theexperimental protocol is described in Example 5. The data is expressedin a bar graph as the specific binding of ¹²⁵I-labelled fibrinogen incpm/well (X 10−3) on the Y-axis against the specific treatments on theX-axis.

FIGS. 10A-10C illustrate leukocyte-endothelium interaction mediated bythe plasma adhesive proteins fibrinogen, vitronectin, and fibronectin.Terminally-differentiated ⁵¹Cr-labeled HL-60 cells were equilibratedwith either fibrinogen, vitronectin, or fibronectin prior to incubationwith resting HUVEC as described in Example 10C1. The number of HL-60cells bound to is plotted in a bar graph on the Y-axis (FIG. 10A).Terminally-differentiated ⁵¹Cr-labeled HL-60 cells were equilibratedwith either vitronectin or transferrin prior to incubation with restingHUVEC for 10 to 60 minutes as described in Example 10C2. The % HL-60cells bound is plotted in a bar graph on the Y-axis (FIG. 10B).Terminally-differentiated ⁵¹Cr-labeled HL-60 cells were equilibratedwith varying concentrations of vitronectin (1.84 to 150 μg/ml) prior toincubation with resting HUVEC as described in Example 10C3. The numbersof bound HL-60 cells is plotted in a bar graph on the Y-axis (FIG. 10C).Data are the mean ± S.D. of triplicates from a representativeexperiment.

FIG. 11 illustrates the γ-chain amino acid residue sequences of humanfibrinogen listed as SEQ ID NO 1. The γ-chain is presented in singleletter amino acid code which corresponds to triple letter amino acidcode in the Sequence Listing. FIG. 11 represents amino acid residue 1 toamino acid residue 411 in the mature protein. The origin of the γ3polypeptide (NNQKIVNLKEKVAQLEA, SEQ ID NO 2) is amino acid residue 117to amino acid residue 133 of the mature γ chain protein. The origin ofthe L10 polypeptide (LGGAKQAGDV, SEQ ID NO 5) is amino acid residue 402to amino acid residue 411 in the mature γ chain protein.

FIG. 12 illustrates the effects of increasing concentrations ofpolypeptide γ3, represented by open diamonds and polypeptide L10,represented by open circles, on the binding of labeled ICAM-1⁺ JYlymphocytes. The assay was performed as described in Example 7B. Thenumber of attached cells is plotted on the Y-axis against peptideconcentration (μg/ml) plotted on the X-axis. The results demonstratethat JY lymphocytes strongly adhered to immobilized γ3 in a specific anddose-dependent fashion, while control peptide L10 did not supportlymphocyte adhesion at any concentration tested under the sameexperimental conditions.

FIG. 13A illustrates the specific attachment of JY lymphocytes to γ3polypeptide or to control L10 polypeptide in solid phase in the presenceof various competitive inhibitors as described in Example 8. Theinhibitors used were monoclonal antibodies 1G12 and 2D5 which recognizeICAM-1 and have been shown to block ICAM-1:fibrinogen interaction,monoclonal antibody 6E6 which recognizes ICAM-1 and has not been shownto block ICAM-1:fibrinogen interaction, and monoclonal antibody 6A11which recognizes EPR-1. The number of attached cells is plotted in a bargraph format. 1G12 and 2D5 competitively inhibited the binding of γ3 toJY lymphocytes thus confirming that γ3 contains the receptor bindingsite of fibrinogen which binds to the ICAM-1 receptor on JY lymphocytes.No binding inhibition occurs with monoclonal antibody 6E6 or 6A11.

FIG. 13BA illustrates the dose-response binding curve of ¹²⁵I-labeled γ3to CHO cells expressing ICAM-1 on their surface. The amount of ¹²⁵I-γ3bound in ng/well is plotted on the Y-axis against peptide concentration(μg/ml) plotted on the X-axis. Specific binding is calculated in thepresence of 100-fold molar excess of unlabelled γ3 or the irrelevantpeptide L10 and is subtracted from the total to calculate the specificbinding depicted. Data are the mean ± standard error of the mean (SEM)of replicates of a representative experiment.

DETAILED DESCRIPTION OF THE INVENTION

A. General Description

The present invention describes the identification of a novel andspecific role for fibrinogen (Fg) in mediating inflammatory processes atendothelial tissues. The role is shown to be a bridging event betweenthe Mac-1 receptor on leukocytes and other Mac-1 receptor-bearing cellsand a class of molecules on endothelial cells referred to herein as anendothelial cell receptor (ECR). The interaction of Fg with the ECR isshown to be a unique RGD-independent binding interaction, different fromthe known binding of Fg to the vitronectin receptor, and to otherendothelial cell surface receptors such as transglutaminase or the 130kilodalton receptor which binds to fibrin-derived peptides.

Insofar as the Fg bridging event described herein adheres Mac-1-bearingcells to endothelial cells, the mechanism discovered and describedherein is distinct from the ICAM-1:Mac-1-dependent pathway of leukocyteadhesion, because of the role played by fibrinogen in the presentbridging interaction. The Fg-dependent inflammation pathway describedherein is referred to as endothelial cell/fibrinogen-mediatedinflammation to emphasize the requirement for fibrinogen in the process.

In a related embodiment, the invention describes a related role forvitronectin (Vn) in mediating inflammatory processes at endothelialtissues. This role is shown herein to be a bridging event betweenleukocytes and endothelial cells. The interaction is shown to be anRGD-independent binding interaction, different from the known binding ofVn to the vitronectin receptor. This Vn-dependent inflammation pathwayis referred to as endothelial cell/vitronectin-mediated inflammation toemphasize the requirement for vitronectin in the process.

B. Homologs

A homolog, as used herein is a macromolecule, typically a protein orpolypeptide, that mimics the structure and function of a domain of aprotein after which it is modeled. Where a native protein carriesmultiple structural domains and thereby mediates multiple distinctfunctions, as with fibrinogen, a homolog mimics a particular domain andis able to interact and compete with the native protein forparticipation with the mediators of that protein function in which thedomain participates.

1. Fibrinogen Homologs

Thus, according to the present invention, a fibrinogen homolog, or Fghomolog, is a macromolecule that mimics a region of fibrinogen thatbinds to an endothelial cell receptor (ECR) in an RGD-independent manneraccording to this invention. The site on ECR to which fibrinogen bindsin an RGD-independent manner is referred to as the “ECR RGD-independentFg-binding site” or “endothelial cell RGD-independent Fg-binding site”,that is, a site on endothelial cells, and on the ECR identified herein,that binds to fibrinogen in an RGD-independent manner. The binding siteis characterized as RGD-independent because endothelial cells containother receptors for binding fibrinogen which mediate binding through theRGD-containing region of fibrinogen.

A Fg homolog is any macromolecule which is capable of binding to the ECRRGD-independent Fg-binding site, and thereby can inhibit Fg binding tothe ECR RGD-independent Fg-binding site on endothelial cells andconsequently inhibit RGD-independent Fg binding to endothelial cells andthe inflammation processes resulting therefrom such binding. Assays formeasuring the binding of a Fg homolog to the ECR RGD-independentFg-binding site are described in Example 3a. Assays for measuring theinhibition of Fg binding to the ECR RGD-independent Fg-binding site aredescribed in Example 5.

A preferred Fg homolog is a fragment of fibrinogen that contains theregion of Fg that binds to the ECR RGD-independent Fg-binding site. Morepreferably, the Fg homolog does not bind to the Mac-1 receptor. Such Fgfragments can be proteolytic fragments of Fg, fibrinogen-derivedpolypeptides, portions of fibrinogen, D₃₀, portions of D₃₀, polypeptidesor proteins homologous to D₃₀ or fibrinogen containing non-natural aminoacid derivatives or non-proteinaceous side chains, analogs or chemicalderivatives of either D₃₀, fibrinogen, fragments or polypeptidesthereof, and conjugates containing a Fg homolog. A preferred Fg homologis fibrinogen, a proteolytic fragment of fibrinogen, and particularlythe D₃₀ fragment of Fg, also referred to as D₃₀.

The D₃₀ fragment of fibrinogen is produced by proteolytic digestion offibrinogen. The preparation of D₃₀ has been -described by Fair et al.,J. Biol. Chem., 256:8018-8023 (1981), Furlaw et al., Biochem. Biophys.Acta., 400:95-11) (1975) and in Example 1. D₃₀ contains partiallydegraded β and γ chains and extensively degraded α chains combined byinter-chain disulfide bonds as described by Pizzo et al., J. Biol.Chem., 247:636-645 (1972). All references and documents cited in thisapplication are hereby incorporated by reference.

The N-terminus of the a chain remnant of D₃₀ originates with amino acidresidues Leu¹³⁶, Gln¹³⁷, Lys¹³⁸ and Asn¹³⁹ using the amino acid sequenceof the alpha chain described by Doolittle et al., Nature, 280:464-469(1979).

The α chain remnant of D₃₀ does not contain amino acid residues Arg⁹⁵,Gly⁹⁶ and Asp⁹⁷ (RGD) or the amino acid residues Arg⁵⁷², Gly⁵⁷³ andAsp⁵⁷⁴ (RGD) and has a Mw of about 11 to 13 kilodaltons (kDa). TheN-termini of the β chain of D₃₀ contains amino acid residues Asp¹³⁴,Asn¹³⁵, Glu¹³⁶, and Asn¹³⁷. The N-terminus of the γ chain of D₃₀contains amino acid residues Met⁸⁹, Leu⁹⁰, Glu⁹¹, and Glu⁹².

Another preferred Fg homolog is a polypeptide derived from fibrinogen,particularly polypeptides having an amino acid residue sequence derivedfrom the ECR binding site on fibrinogen in D₃₀ as described herein.Particularly preferred are polypeptides that include an amino acidresidue sequence of the fibrinogen (Fg) γ chain from residues 117 to 133of the γ chain. The Fg γ chain sequence is shown in SEQ ID NO 1, and theregion of γ chain from residues 117 to 133 is shown in SEQ ID NO 2.

Preferred Fg homolog polypeptides are short amino acid residue sequencesfor ease of synthesis, manipulation, storage and versatility of usebased on their specific biological properties. In particular, smallpolypeptides are able to contain (mimic) one but not another bindingspecificity of Fg, thereby increasing the selectivity and consequentutility of the reagent. A preferred polypeptide has a total sequencelength of from about 17 to about 100 amino acids.

Particularly preferred polypeptides are described in the Examples, andinclude the 17 residue polypeptide corresponding to residues 117-133 ofFg γ chain, and variants of this polypeptide which preserve the basicactivity described herein. Terminus-modified polypeptides are describedhaving added residues at the amino and/or carboxy termini. These includethe addition of a cysteine (C) residue to the carboxy terminus, shown inSEQ ID NO 3, and the addition of a tripeptide lysine-tyrosine-glycine(KYG) to the amino terminus, shown in SEQ ID NO 4. Thus preferredpolypeptides include, and preferably consist essentially of, an aminoacid residue sequence selected from the group consisting of SEQ ID NOs,2, 3 and 4.

A subject Fg homolog includes any analog, fragment or chemicalderivative of an active fibrinogen polypeptide as defined herein, solong as the polypeptide is capable of inhibiting Fg binding to the ECRRGD-independent Fg-binding site. Therefore, a polypeptide Fg homolog canbe subject to various changes, substitutions, insertions, modificationand deletions where such changes provide for certain advantages in itsuse, which changes are summarily referred to as “variants thereof”. Inthis regard, a Fg homolog of this invention can contain one or morechanges in the polypeptide so long as the homolog retains its functionin one or more of the binding and inhibition assays as defined herein.

The term “analog” includes any polypeptide having an amino acid residuesequence substantially identical to a sequence of a Fg homolog or domainof fibrinogen in which one or more residues have been conservativelysubstituted with a functionally similar residue and which displays theabilities as described herein. Examples of conservative substitutionsinclude the substitution of one non-polar (hydrophobic) residue such asisoleucine, valine, leucine or methionine for another, the substitutionof one polar (hydrophilic) residue for another such as between arginineand lysine, between glutamine and asparagine, between glycine andserine, the substitution of one basic residue such as lysine, arginineor histidine for another, or the substitution of one acidic residue,such as aspartic acid or glutamic acid for another.

The phrase “conservative substitution” also includes the use of achemically derivatized residue in place of a non-derivatized residueprovided that such polypeptide displays the requisite binding activity.

“Chemical derivative” refers to a polypeptide having one or moreresidues chemically derivatized by reaction of a functional side group.Such derivatized molecules include for example, those molecules in whichfree amino groups have been derivatized to form amine hydrochlorides,p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonylgroups, chloroacetyl groups or formyl groups. Free carboxyl groups maybe derivatized to form salts, methyl and ethyl esters or other types ofesters or hydrazides. Free hydroxyl groups may be derivatized to form0-acyl or 0-alkyl derivatives. The imidazole nitrogen of histidine maybe derivatized to form N-im-benzylhistidine. Also included as chemicalderivatives are those peptides which contain one or more naturallyoccurring amino acid derivatives of the twenty standard amino acids. Forexample: 4-hydroxyproline may be substituted for proline;5-hydroxylysine may be substituted for lysine; 3-methylhistidine may besubstituted for histidine; homoserine may be substituted or serine; andornithine may be substituted for lysine. Polypeptides of the presentinvention also include any polypeptide having one or more additionsand/or deletions or residues relative to the sequence of a polypeptidewhose sequence is shown herein, so long as the requisite bindingactivity is maintained.

The term “fragment” refers to any subject polypeptide having an aminoacid residue sequence shorter than that the native protein.

When a polypeptide defining a portion of a Fg homolog of the presentinvention has a sequence that is not identical to the sequence of aportion of fibrinogen, it is typically because one or more conservativeor non-conservative substitutions have been made, usually no more thanabout 30 number percent, more usually no more than 20 number percent,and preferably no more than 10 number percent of the amino acid residuesare substituted. Additional residues may also be added at eitherterminus for the purpose of providing a “linker” by which thepolypeptides of this invention can be conveniently affixed to a label orsolid matrix, or carrier. Preferably the linker residues do not form Fghomolog epitopes, i.e., are not similar in structure to a Fg homolog.

Labels, solid matrices and carriers that can be used with the Fghomologs of this invention are described hereinbelow.

Amino acid residue linkers are usually at least one residue and can be40 or more residues, more often 1 to 10 residues, but do not form Fgepitopes. Typical amino acid residues used for linking are tyrosine,cysteine, lysine, glutamic and aspartic acid, or the like. In addition,a subject polypeptide can differ, unless otherwise specified, from thenatural sequence of the γ chain of fibrinogen by the sequence beingmodified by terminal-NH₂ acylation, e.g., acetylation, or thioglycolicacid amidation, by terminal-carboxylamidation, e.g., with ammonia,methylamine, and the like.

A polypeptide forming a Fg homolog of the present invention can beprepared using the solid-phase synthetic technique initially describedby Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963). Other polypeptidesynthesis techniques may be found, for example, in M. Bodanszky et al.,Peptide Synthesis, John Wiley & Sons, 2d Ed., (1976) as well as in otherreference works known to those skilled in the art. A summary ofpolypeptide synthesis techniques may be found in J. Stuart and J. D.Young, Solid Phase Peptide Synthesis, Pierce Chemical Company, Rockford,Ill., 3d Ed., Neurath, H. et al., Eds., p. 104-237, Academic Press, NewYork, N.Y. (1976). Appropriate protective groups for use in suchsyntheses will be found in the above texts as well as in J. F. W.McOmie, Protective Groups in Organic Chemistry, Plenum Press, New York,N.Y. (1973).

In general, those synthetic methods comprise the sequential addition ofone or more amino acid residues or suitably protected amino acidresidues to a growing polypeptide chain. Normally, either the amino orcarboxyl group of the first amino acid residue is protected by asuitable, selectively removable protecting group is utilized for aminoacids containing a reactive side group such as lysine.

Using a solid phase synthesis as an example, the protected orderivatized amino acid is attached to an inert solid support through itsunprotected carboxyl or amino group. The protecting group of the aminoor carboxyl group is then selectively removed and the next amino acid inthe sequence having the complementary (amino or carboxyl) group suitablyprotected is admixed and reacted under conditions suitable for formingthe amid linkage with the residue already attached to the solid support.The protecting group of the amino or carboxyl group is then removed fromthis newly added amino acid residue, and the next amino acid (suitablyprotected) is then added, and so forth. After all the desired aminoacids have been linked in the proper sequence any remaining terminal andside group protecting groups (and solid support) are removedsequentially or concurrently, to provide the final polypeptide. Apreferred polypeptide synthesis method is described in the Examples.

Methods and procedures for determining inhibition are well known in theart and include the use of competition assays similar to the antigencompetition assays described in Antibodies: A Laboratory Manual, Harlowand Lane, eds., Cold Spring Harbor, N.Y. (1988). For example, anunlabelled compound suspected of being a Fg homolog can be used toinhibit the binding of labelled Fg or a labelled Fg homolog to the ECRRGD-independent Fg-binding site of ECR on endothelial cells. The amountof labelled Fg binding to the binding site in the presence or absence ofthe unlabelled compound would be compared and if the presence of theunlabelled compound inhibits the amount of labelled Fg binding to thebinding site, then the unlabelled compound is a Fg homolog. A preferredmethod for measuring inhibition of Fg binding to ECR is described inExample 5.

Another Fg homolog contemplated by the present invention are antibodymolecules which immunoreact with the ECR RGD-independent Fg-bindingsite. Exemplary antibody molecules are anti-ECR antibodies andanti-ICAM-1 antibodies, defined further herein.

A further Fg homolog contemplated by the present invention is anybifunctional molecule which contains a Fg homolog of this invention, andtherefore possesses the biological activity, and uses therefor, of a Fghomolog of this invention. A bifunctional Fg homolog molecule can be,for example, a polypeptide which contains two amino acid residuesequences operatively linked into a single polypeptide that defines botha Fg homolog and another polypeptide function. A preferred bifunctionalpolypeptide defines both the ECR-binding site on fibrinogen and theMac-1 binding site on fibrinogen, referred to as an ECR/Mac-1 bindingpolypeptide.

An ECR/Mac-1 binding polypeptide comprises the amino acid residuesequence defining the ECR binding site, i.e., the Fg γ chain sequencefrom residues 117 to 133, and further contains the amino acid residuesequence defining the Mac-1 binding site, i.e., the Fg γ chain sequencefrom residues 190 to 202. A preferred ECR/Mac-1 binding polypeptide hasthe amino acid residue sequence that comprises residues 117 to 202 offibrinogen γ.

Alternatively, a bifunctional Fg homolog can be formed using fusionpolypeptide technology, so long as the included functional regionsinclude residues 117-133, and residues 190-202.

2. Endothelial Cell Receptor Homologs

An endothelial cell receptor (ECR) homolog, according to the presentinvention, is a macromolecule that mimics a region of ECR that binds tofibrinogen in an RGD-independent manner. The site on fibrinogen to whichECR binds in an RGD-independent manner is referred to as the “FgRGD-independent ECR-binding site”, that is, a site on Fg that binds toECR in an RGD-independent manner.

An ECR homolog is any macromolecule which is capable of binding to theFg RGD-independent ECR-binding site, and thereby can inhibit ECR bindingto the Fg RGD-independent ECR-binding site on Fg, and thereby inhibitRGD-independent Fg binding to endothelial cells. Assays for measuringthe binding of an ECR homolog to the Fg RGD-independent ECR-binding siteare described in Example 3A. Assays for measuring the inhibition of ECRbinding to the Fg RGD-independent ECR-binding site are described inExample 5.

A preferred ECR homolog is a substantially purified ECR protein, or afragment of ECR that contains a region of ECR that binds to the FgRGD-independent ECR-binding site. A particularly preferred ECR homologis the ECR protein identified as ICAM-1 in Example 2. Thus, where thespecies of ECR is ICAM-1, the ECR homolog is referred to herein as anICAM-1 homolog. Also contemplated ECR homologs are ICAM-1 associated orICAM-1 related molecules.

An ECR homolog of the present invention may be modified, fragmented,coupled or otherwise manipulated as described herein for a Fg homolog solong as the desirable binding and inhibiting properties are maintained.

Particularly preferred are polypeptides and their analogues derived fromthe region of ICAM-1 that binds to fibrinogen, particularly a solubleICAM-1 and functional derivatives thereof. A soluble ICAM-1 for use in acomposition or method described herein typically lacks the transmembranedomain that serves as a membrane anchor. ICAM-1 and soluble ICAM-1compositions suitable for use as an ECR homolog can be prepared by avariety of methods, including the purification methods described inExample 2. Additional preparative methods for ICAM-1 and soluble ICAM-1are described in the Published EPO Application No. EP 365837 andPublished Canadian Application No. 2008368.

Another ECR homolog contemplated by the present invention is an antibodymolecule which immunoreacts with the Fg RGD-independent ECR-bindingsite. Exemplary antibody molecules are anti-Fg antibodies, definedfurther herein.

A Fg or ECR homolog can be coupled to or conjugated with another proteinor polypeptide to produce a homolog conjugate. A homolog conjugate hasadvantages over a homolog used alone. For example, coupling the homologto a protein or polypeptide known to contain a second biologicalfunction allows the targeting of that second biological function to thelocation at or near an ECR.

When coupled to a carrier to form what is known in the art as acarrier-hapten conjugate, a homolog of the present invention is capableof inducing antibodies that immunoreact with the homolog. In view of thewell established principle of immunologic cross-reactivity, the presentinvention therefore contemplates antigenically related variants of a Fgor ECR homolog. An “antigenically related variant” is a subjectpolypeptide that is capable of inducing antibody molecules thatimmunoreact with a Fg homolog of this invention and with Fg, or with anECR homolog of this invention and with ECR.

Any peptide of the present invention may be used in the form of apharmaceutically acceptable salt. Suitable acids which are capable offorming salts with the peptides of the present invention includeinorganic acids such as hydrochloric acid, hydrobromic acid, perchloricacid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric aceticacid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalicacid, malonic acid, succinic acid, maleic acid, fumaric acid,anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilicacid or the like.

Suitable bases capable of forming salts with the peptides of the presentinvention include inorganic bases such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide and the like; and organic bases such asmono-, di- and tri-alkyl and aryl amines (e.g. triethylamine,diisopropyl amine, methyl amine, dimethyl amine and the like) andoptionally substituted ethanolamines (e.g. ethanolamine, diethanolamineand the like).

In other preferred embodiments a homolog is conjugated with a carriermolecule to form a homolog conjugate containing at least one carriermolecule. Typical carriers include Sepharose™, Sephadex™, proteins,polypeptides and the like.

A homolog may also be conjugated to itself or aggregated in such a wayas to produce a large complex containing a homolog. A large complexcontaining a homolog is advantageous because it has new biologicproperties such as longer half-life in circulation or greater activity.

3. Vitronectin Homologs

A vitronectin (Vn) homolog, according to the present invention, is amacromolecule that mimics a region of Vn that is capable of binding toendothelial cells in an RGD-independent manner, and in doing so competeswith the ability of Vn to mediate leukocyte adhesion onto endothelialcells. The site on endothelial cells to which Vn binds in thisembodiment is referred to as an “endothelial cell RGD-independentVn-binding site”, that is, a site on endothelial cells that binds to Vnin an RGD-independent manner.

A Vn homolog is any macromolecule which is capable of binding to theendothelial cell RGD-independent Vn-binding site, and thereby caninhibit Vn binding to the endothelial cell RGD-independent Vn-bindingsite and consequently inhibit RGD-independent Vn-mediated adhesion ofleukocytes onto endothelial cells and the inflammation processesresulting therefrom such binding. Assays for measuring the binding of aVn homolog to the endothelial cell RGD-independent Vn-binding site aredescribed in the Examples. Assays for measuring the inhibition of Vnbinding to the endothelial cell RGD-independent Vn-binding site are alsodescribed in the Examples.

A preferred Vn homolog is a fragment of vitronectin that contains theregion of Vn that binds to the endothelial cell RGD-independentFg-binding site. Such Vn fragments can be proteolytic fragments of Vn,vitronectin-derived polypeptides and portions of vitronectin.

C. Compositions Containing Homologs

In one preferred embodiment, the invention contemplates a compositioncomprising a carrier and a fibrinogen (Fg) homolog according to thisinvention capable of binding to the ECR RGD-independent Fg-binding siteand inhibiting fibrinogen binding to the ECR RGD-independent Fg-bindingsite. Preferred Fg homologs for use in a composition were describedearlier. A particularly preferred Fg homolog for use in a composition isa Fg homolog polypeptide of this invention.

In a related embodiment, the invention contemplates a compositioncomprising a carrier and an endothelial cell receptor homolog accordingto this invention capable of binding to the Fg RGD-independentECR-binding site and inhibiting ECR binding to the Fg RGD-independentECR-binding site. Preferred ECR homologs, particularly ICAM-1 homologs,for use is a composition were described earlier.

Both of these compositions are useful for inhibiting the binding of Fgto endothelial cells. When practiced in vivo, inhibiting Fg binding toendothelial cells inhibits endothelial cell/fibrinogen-mediatedinflammation and the associated disease processes described in moredetail elsewhere herein.

The Fg or ECR homolog is typically present in an effective amount, thatis, an amount sufficient to be used to inhibit Fg binding to endothelialcells, or an Fg-binding inhibiting amount. Assays for determiningeffective amounts of a homolog are readily available, such as thosedescribed herein, and can be used to determine effective amounts.

The homolog is preferably present in the composition in substantiallypure form. Furthermore, the homolog is typically in a physiologicallyacceptable composition, i.e., a composition compatible with endothelialcells. When formulated for use in vivo, the composition is typicallypharmaceutically acceptable, and the amount is referred to as atherapeutically effective amount.

A therapeutically effective amount of a Fg or ECR homolog is an amountthat when administered to a patient is capable of inhibiting fibrinogenbinding to endothelial cells. Assays for detecting the inhibition of Fgbinding to endothelial cells and thereby measuring effective inhibitingamounts of homolog include, but are not limited to the competitive andother binding assays described in Example 5 of this specification.

Preferably, a therapeutically effective amount of a Fg or ECR homolog isan amount that reduces (inhibits) fibrinogen binding to endothelialcells by at least 10 percent, preferably by at least 50 percent, andmore preferably by at least 99 percent, when measured in an in vitroassay for fibrinogen binding to endothelial cells. An exemplary in vitroassay to quantitate effective inhibitory amounts of a Fg homolog isdescribed in Example 5. Typically, a composition of this inventioncontains at least about 0.1 weight percent of homolog in the totalweight of the composition.

In one embodiment, a therapeutic composition is useful for inhibitingendothelial cell/fibrinogen mediated inflammation in a patientexhibiting one or more of the conditions associated with inflammation asdescribed further herein. In this embodiment, a therapeuticallyeffective amount is an amount that when administered to a patient issufficient to inhibiting fibrinogen binding to endothelial cell, therebyinhibiting endothelial cell/fibrinogen mediated inflammation.

Assays for directly detecting the inhibition of endothelialcell/fibrinogen mediated inflammation include, but are not limited to,clinical inspection of symptoms attendant in a patient presenting withinflammation.

Substantially pure, when used in the context of a Fg or ECR homolog,refers to compositions that are enriched in Fg or ECR homolog, andpreferably are free of detectable amounts of blood cells, immunoglobulinand albumin proteins, and lipoproteins, and more preferably contains inexcess of 99 percent by weight of homolog per total mass in thecomposition.

In one embodiment, the present invention contemplates compositions, andtheir methods of use, comprising both an Fg and ECR homolog of thisinvention in a range of ratios of Fg homolog to ECR homolog. The ratiocan be anywhere from vast excesses of Fg homolog relative to ECRhomolog, to vast excesses of ECR homolog to Fg homolog, ie, about0.01:99.00 percent by weight. Preferred ratios range from 10:1 to 1:1.The only impermissible combination of Fg homologs and ECR homologs in acontemplated composition is the admixture of an anti-ECR antibody (Fghomolog) with an ECR homolog, or the admixture of an anti-Fg antibody(ECR homolog) with a Fg homolog, because these particular combinationswill cross-immunoreact and neutralize effectiveness.

By pharmaceutically acceptable is meant that a Fg or ECR homolog, whenused in a therapeutic composition, does not cause any undesirablephysiological effects due to the presence of contaminants. Thus apharmaceutically acceptable Fg or ECR homolog is free ofpharmaceutically unacceptable contaminants such as pyrogens(lipopolysaccharides) and other contaminants such as poisonous chemicals(i.e., sodium azide) and detergents, namely sodium dodecyl sulfate.

The preparation of therapeutic compositions which contain polypeptidesor proteins as active ingredients is well understood in the art.Typically, such compositions are prepared as injectables, either asliquid solutions or suspensions, however, solid forms suitable forsolution in, or suspension in, liquid prior to injection can also beprepared. The preparation can also be emulsified. The active therapeuticingredient is mixed with (dispersed in) inorganic and/or organiccarriers which are pharmaceutically acceptable and compatible with theactive ingredient. Carriers are pharmaceutically acceptable excipients(vehicles) comprising more or less inert substances when added to atherapeutic composition to confer suitable consistency or form to thecomposition. Suitable carriers are, for example, water, saline,dextrose, glycerol, ethanol, or the like and combinations thereof. Inaddition, if desired, the composition can contain minor amounts ofauxiliary substances such as wetting or emulsifying agents and pHbuffering agents which enhance the effectiveness of the activeingredient.

A therapeutic composition useful in the practice of the presentinvention typically contains a Fg or ECR homolog formulated into thetherapeutic composition as a neutralized pharmaceutically acceptablesalt form. Pharmaceutically acceptable salts include the acid additionsalts (formed with the free amino groups of the polypeptide or antibodymolecule) and which are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric, mandelic, and the like. Salts formed with thefree carboxyl groups can also be derived from inorganic bases such as,for example, sodium, potassium, ammonium, calcium, or ferric hydroxides,and such organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

The therapeutic Fg or ECR homolog-containing composition isconventionally administered parenterally, as by injection of a unitdose, for example. In this way the therapeutic composition can bedelivered by a variety of means including intravenous, intramuscular,infusion, oral, intranasal, intraperitoneal, subcutaneous, rectal,topical, or into other regions, such as into synovial fluids. Howeverdelivery of a Fg or ECR homolog-containing composition transdermally isalso contemplated, such by diffusion via a transdermal patch.

The term “unit dose” when used in reference to a therapeutic compositionused in the present invention refers to physically discrete unitssuitable as unitary dosages for humans, each unit containing apredetermined quantity of active material calculated to produce thedesired therapeutic effect in association with the required diluent,carrier or excipient.

In preferred embodiments, a therapeutic composition of the presentinvention contains an effective amount of a Fg or ECR homolog and isprepared by dispersing the homolog in a sterile solution to form asterile composition. A sterile composition is well understood in thepharmaceutical arts and is substantially free of microorganisms such asa virus, bacteria or fungus. Typically a composition is made sterile bypassing the composition through a filter such as a 0.2 micron filterdesigned for this purpose.

In other preferred embodiments, a composition of the present inventionis optimized to allow the Fg or ECR homolog it contains to be deliveredtransdermally.

In other preferred embodiments, a composition of the present inventioncontains an immunologically effective amount of Fg or ECR homolog. Animmunologically effective amount of Fg or ECR homolog is an amountsufficient to produce antibodies that immunoreact with the immunizedhomolog.

D. Antibodies and Monoclonal Antibodies

The term “antibody” in its various grammatical forms is used herein as acollective noun that refers to a population of immunoglobulin moleculesand/or immunologically active portions of immunoglobulin molecules,i.e., molecules that contain an antibody combining site or paratope.

An “antibody combining site” is that structural portion of an antibodymolecule comprised of the heavy and light chain variable andhypervariable regions that specifically binds antigen. The site can bereduced to a minimum amount of the complementarity determining regions(CDRs) of the variable regions so long as the antigen-binding propertyis preserved.

The phrase “antibody molecule” in its various grammatical forms as usedherein therefore contemplates both an intact immunoglobulin molecule andan immunologically active portion of an immunoglobulin molecule, i.e.,an immunoreactive fragment of an intact immunoglobulin molecule.

Exemplary antibody molecules for use in the methods and systems of thepresent invention are intact immunoglobulin molecules, substantiallyintact immunoglobulin molecules and those portions of an immunoglobulinmolecule that contain the paratope, including those portions known inthe art as Fab, Fab′, F(ab′)₂, F(v), and portions thereof.

Fab and F(ab′)₂ portions of antibodies can be prepared by theproteolytic reaction of papain and pepsin, respectively, onsubstantially intact antibodies by methods that are well known. See forexample, U.S. Pat. No. 4,342,566, Inbar et al., Proc. Natl. Acad. Sci.USA, 69:2659-62 (1972), and Goding, Monoclonal Antibodies: Principlesand Practice, Academic Press, P118-124 (1983).

Fab′ antibody portions are also well known and are produced from F(ab′)₂portions followed by reduction of the disulfide bonds linking the twoheavy reduction of the disulfide bonds linking the two heavy chainportions as with mercaptoethanol, and followed by alkylation of theresulting protein mercaptan with a reagent such as iodoacetamide. Anantibody containing intact antibody molecules are preferred, and areutilized as illustrative herein.

1. Anti-ECR Homolog Antibodies

In one embodiment, the present invention contemplates an anti-ECRhomolog antibody, ie, a composition comprising antibody molecules, thatimmunoreacts with an ECR homolog of this invention at or near the ECRRGD-independent Fg-binding site. Stated differently, and anti-ECRhomolog antibody immunoreacts with ECR, is specific for the Fg-bindingsite on ECR as defined herein and inhibits fibrinogen binding toendothelial cells. A preferred anti-ECR homolog antibody preferentiallyinhibits fibrinogen binding to stimulated endothelial cells.

A preferred anti-ECR antibody does not immunoreact with vitronectinreceptor (VnR), ie, is substantially free of antibody molecules thatimmunoreact with VnR. More preferably, the antibody does not immunoreactwith any RGD-dependent endothelial cell surface receptor, or withtransglutaminase or the 130 kilodalton receptor which binds fibrinpeptides.

By “preferentially inhibits” is meant that the antibody exhibits moreinhibition of Fg binding to endothelial cells that are stimulated thanendothelial cells that are not stimulated when assayed on endothelialcells in monolayer, such as is described in Example 5. In other words, apreferred antibody inhibits Fg binding to stimulated endothelial cellsat lower antibody doses or resulting in lower Fg binding than the sameantibody when tested on non-stimulated endothelial cells. Endothelialcells may be stimulated by a variety of means, including by exposure tocytokines such as TNF and chemoattractant peptides such as N-FMLP.

2. Anti-Fg Homolog Antibodies

In one embodiment, the invention describes an anti-fibrinogen homologantibody molecule that immunoreacts with fibrinogen or a fibrinogenhomolog of this invention.

Preferred anti-Fg homolog antibodies immunoreact with a Fg homologpolypeptide of this invention. Exemplary polypeptides are thepolypeptides described earlier having a sequence shown in SEQ ID NOs 2,3 or 4.

Particularly preferred are anti-Fg homolog antibodies which areimmunospecific for the site on Fg that binds to ECR in an RGDindependent manner, i.e., the Fg RGD-independent ECR-binding sitedescribed herein. This class of antibody specificity is preferredbecause it allows for the diagnostic and therapeutic advantagesdescribed herein for reagents having the selective binding propertiesrelated to the Fg RGD-independent ECR-binding site. Thus, a preferredantibody is substantially free of immunoreactivity with otherfunctionally separate sites on fibrinogen, such as the Mac-1 bindingsite.

The Mac-1 binding site on fibrinogen has been defined by fibrinogenpolypeptides as described by Altieri et al in published InternationalPCT Application No. PCT/US91/05096. A polypeptide corresponding toresidues 190-202 of the Fg γ chain, and shown at residues 190-202 of SEQID NO 1, inhibits D₃₀ binding to monocytes, was shown to define theMac-1 binding site. Therefore, a preferred anti-Fg homolog antibody ofthis invention does not immunoreact with the polypeptide shown in SEQ IDNO 1 from residues 190 to 202.

By “substantially free” means that an antibody molecule of thisinvention does not immunoreact with the stated antigen at levels withinone order of magnitude, and preferably within two orders of magnitude,of the level of immunoreaction with a species of antigen recited toimmunoreact with the antibody molecule when immunoreaction is expressedas an equilibrium constant between bound (immunoreacted) and nonboundantigen.

Antibody reactivity with a stated antigen can be measured by a varietyof immunological assays known in the art. Exemplary immunoreactionassays are described herein.

The preparation of antibodies is well known in the art. See, Staudt etal., J. Exp. Med., 157:687-704 (1983), Examples 4 and 6 of thespecification or Antibodies: A Laboratory Manual, Harlowe and Lane,Eds., Cold Spring Harbor, N.Y. (1988). Briefly, to produce an antibodycomposition of this invention, a laboratory mammal is inoculated with animmunologically effective amount of a ECR homolog, typically as presentin a vaccine or inoculum of the present invention, thereby inducing inthe mammal antibody molecules having immunospecificity for theimmunogen. The antibody molecules induced are then collected from themammal and are isolated to the extent desired by well known techniquessuch as, for example, by immunoaffinity chromatography, or by using DEAESephadex™ to obtain the IgG fraction.

To enhance the specificity of the antibody, the antibody molecules arepreferably purified by immunoaffinity chromatography using solidphase-affixed immunogen. The antibody is contacted with the solidphase-affixed immunogen for a period of time sufficient for theimmunogen to immunoreact with the antibody molecules to form a solidphase-affixed immunocomplex. The bound antibodies are separated from thecomplex by standard techniques.

A vaccine useful for preparing antibodies of the present inventioncomprises immunologically effective amounts of both an immunogen and animmunopotentiator suitable for use in mammals.

An immunopotentiator is a molecular entity that stimulates maturation,differentiation and function of B and/or T lymphocytes.Immunopotentiators are well known in the art and include T cellstimulating polypeptides such as those described in U.S. Pat. No.4,426,324 and the C8-substituted guanine nucleosides described byGoodman et al., J. Immunol., 135:3284-88 (1985) and U.S. Pat. No.4,643,992.

The word “inoculum” in its various grammatical forms is used herein todescribe a composition containing, for example, a Fg homolog or an ECRhomolog of this invention as an active ingredient used for thepreparation of antibodies of this invention.

When a small molecule such as a polypeptide is used in an inoculum toinduce antibodies it is to be understood that the polypeptide can beused in various embodiments, e.g., alone or linked to a carrier as aconjugate, or as a polypeptide polymer. However, for ease of expressionand in context of a polypeptide inoculum, the various embodiments of thepolypeptides of this invention are collectively referred to herein bythe term “polypeptide” and its various grammatical forms.

For a polypeptide that contains fewer than about 35 amino acid residues,it is preferable to use the peptide bound to a carrier for the purposeof inducing the production of antibodies.

One or more additional amino acid residues can be added to the amino- orcarboxy-termini of the polypeptide to assist in binding the polypeptideto a carrier. Cysteine residues added at the amino- or carboxy-terminiof the polypeptide have been found to be particularly useful for formingconjugates via disulfide bonds. However, other methods well known in theart for preparing conjugates can also be used. An exemplary polypeptidehaving a cysteine residue at its carboxy terminus for conjugation to acarrier is shown in SEQ ID NO 3.

The techniques of polypeptide conjugation or coupling through activatedfunctional groups presently known in the art are particularlyapplicable. See, for example, Aurameas, et al., Scand. J. Immunol., Vol.8, Suppl. 7:7-23 (1978) and U.S. Pat. No. 4,493,795, No. 3,791,932 andNo. 3,839,153. In addition, a site directed coupling reaction can becarried out so that any loss of activity due to polypeptide orientationafter coupling can be minimized. See, for example, Rodwell et al.,Biotech., 3:889-894 (1985), and U.S. Pat. No. 4,671,958.

Exemplary additional linking procedures include the use of Michaeladdition reaction products, di-aldehydes such as glutaraldehyde,Klipstein, et al., J. Infect. Dis., 147:318-326 (1983) and the like, orthe use of carbodiimide technology as in the use of a water-solublecarbodiimide to form amide links to the carrier. Alternatively, theheterobifunctional cross-linker SPDP (N-succinimidyl-3-(2-pyridyldithio)proprionate)) can be used to conjugate peptides, in which acarboxy-terminal cysteine has been introduced.

Useful carriers are well known in the art, and are generally proteinsthemselves. Exemplary of such carriers are keyhole limpet hemocyanin(KLH), edestin, thyroglobulin, albumins such as bovine serum albumin(BSA) or human serum albumin (HSA), red blood cells such as sheeperythrocytes (SRBC), tetanus toxoid, cholera toxoid as well as polyaminoacids such as poly (D-lysine: D-glutamic acid), and the like.

The choice of carrier is more dependent upon the ultimate use of theinoculum and is based upon criteria not particularly involved in thepresent invention. For example, a carrier that does not generate anuntoward reaction in the particular animal to be inoculated should beselected.

The present inoculum contains an effective, immunogenic amount of ahomolog of this invention, typically as a conjugate linked to a carrier.The effective amount of homolog per unit dose sufficient to induce animmune response to the immunogen depends, among other things, on thespecies of animal inoculated, the body weight of the animal and thechosen inoculation regimen as is well known in the art. Inoculatypically contain homolog concentrations of about 10 micrograms to about500 milligrams per inoculation (dose), preferably about 50 micrograms toabout 50 milligrams per dose.

The term “unit dose” as it pertains to the inocula refers to physicallydiscrete units suitable as unitary dosages for animals, each unitcontaining a predetermined quantity of active material calculated toproduce the desired immunogenic effect in association with the requireddiluent; i.e., carrier, or vehicle. The specifications for the novelunit dose of an inoculum of this invention are dictated by and aredirectly dependent on (a) the unique characteristics of the activematerial and the particular immunologic effect to be achieved, and (b)the limitations inherent in the art of compounding such active materialfor immunologic use in animals, as disclosed in detail herein, thesebeing features of the present invention.

Inocula are typically prepared from the dried solid homolog-conjugate bydispersing the conjugate in a physiologically tolerable (acceptable)diluent such as water, saline or phosphate-buffered saline to form anaqueous composition.

Inocula can also include an adjuvant as part of the diluent. Adjuvantssuch as complete Freund's adjuvant (CFA), incomplete Freund's adjuvant(IFA) and alum are materials well known in the art, and are availablecommercially from several sources.

The anti-homolog specific antibody so produced can be used, inter alia,in the therapeutic and diagnostic methods and systems of the presentinvention to inhibit fibrinogen binding to endothelial cells, and todetect homologs present in a sample such as a body fluid sample. Inparticular, the antibodies can be used to monitor the therapeutic fateand half-life of homologs administered according to the therapeuticmethods of the invention.

An antibody of this invention is preferably a monoclonal antibody due tothe controlled specificity offered by a monoclonal antibody.

The phrase “monoclonal antibody” in its various grammatical forms refersto a population of antibody molecules that contain only one species ofantibody combining site capable of immunoreacting with a particularepitope. A monoclonal antibody thus typically displays a single bindingaffinity for any epitope with which it immunoreacts. A monoclonalantibody may therefore contain an antibody molecule having a pluralityof antibody combining sites, each immunospecific for a differentepitope, e.g., a bispecific monoclonal antibody.

A monoclonal antibody is typically composed of antibodies produced byclones of a single cell called a hybridoma that secretes (produces) onlyone kind of antibody molecule. The hybridoma cell is formed by fusing anantibody-producing cell and a myeloma or other self-perpetuating cellline.

The preparation of such antibodies was first described by Kohler andMilstein, Nature 256:495-497 (1975). An exemplary hybridoma technologyis described by Niman et al., Proc. Natl. Acad. Sci., U.S.A.,80:4949-4953 (1983). Other methods of producing a monoclonal antibody, ahybridoma cell, or a hybridoma cell culture are also well known. See,for example, Antibodies: A Laboratory Manual, Harlow et al., Cold SpringHarbor Laboratory, 1988; or the method of isolating monoclonalantibodies from an immunological repertoire as described by Sastry, etal., Proc. Natl. Acad. Sci. USA, 86:5728-5732 (1989); and Huse et al.,Science, 246:1275-1281 (1981). The references cited are herebyincorporated herein by reference.

The hybridoma so prepared produces a supernate that can be screened forthe presence of antibody molecules that immunoreact with a homolog ofthis invention, or for inhibition of fibrinogen binding to endothelialcells as described further herein.

Briefly, to form the hybridoma from which the monoclonal antibodycomposition is produced, a myeloma or other self-perpetuating cell lineis fused with lymphocytes obtained from the spleen of a mammalhyperimmunized with a homolog of this invention as the immunogen.

It is preferred that the myeloma cell line used to prepare a hybridomabe from the same species as the lymphocytes. Typically, a mouse of thestrain 129 GlX⁺ is the preferred mammal. Suitable mouse myelomas for usein the present invention include thehypoxanthine-aminopterin-thymidine-sensitive (HAT) cell linesP3X63-Ag8.653, and Sp2/0-Ag14 that are available from the American TypeCulture Collection, Rockville, Md., under the designations CRL 1580 andCRL 1581, respectively.

Splenocytes are typically fused with myeloma cells using polyethyleneglycol (PEG) 1500. Fused hybrids are selected by their sensitivity toHAT. Hybridomas producing a monoclonal antibody of this invention areidentified using the enzyme linked immunosorbent assay (ELISA) describedin Example 4.

A monoclonal antibody of the present invention can also be produced byinitiating a monoclonal hybridoma culture comprising a nutrient mediumcontaining a hybridoma that produces and secretes antibody molecules ofthe appropriate polypeptide specificity. The culture is maintained underconditions and for a time period sufficient for the hybridoma to secretethe antibody molecules into the medium. The antibody-containing mediumis then collected. The antibody molecules can then be further isolatedby well known techniques.

Media useful for the preparation of these compositions are both wellknown in the art and commercially available and include syntheticculture media, inbred mice and the like. An exemplary synthetic mediumis Dulbecco's minimal essential medium (DMEM; Dulbecco et al., Virol.8:396 (1959)) supplemented with 4.5 gm/1 glucose, 20 mm glutamine, and20% fetal calf serum. An exemplary inbred mouse strain is the BALB/c.

The monoclonal antibodies of this invention can be used in the samemanner as disclosed herein for antibodies of the present invention.

For example, the monoclonal antibody can be used in the therapeutic,diagnostic or in vitro methods disclosed herein where inhibition offibrinogen binding to endothelial cells is desired.

A preferred monoclonal antibody immunoreacts with the prototype ECRdescribed herein, namely ICAM-1. The anti-ICAM-1 monoclonal antibody wasproduced by immunization with endothelial cells, followed by screeningfor the ability to inhibit Fg binding to endothelial cells, as describedin Example 4.

A particularly preferred anti-ICAM-1 monoclonal antibody produced usingendothelial cells as the immunogen is the monoclonal antibody producedby the hybridoma 14E11, 16G8, 2E12, or 2B12 that immunoreact withICAM-1, and inhibit Fg binding to endothelial cells.

Additional anti-ICAM-1 monoclonal antibodies were produced byimmunization with Daudi cells, followed by screening for the ability tofirst immunoreact with endothelial cells, and further screened for theability to inhibit leukocyte adhesion to HUVEC cells in the presence ofFg binding as described in Example 7.

A particularly preferred anti-ICAM-1 monoclonal antibody produced usingDaudi cells as the immunogen is the monoclonal antibody produced by thehybridoma 1G12 or 2D5 that immunoreact with ICAM-1, and are shown hereinto inhibit Fg binding to endothelial cells.

Also contemplated are monoclonal antibodies having a binding specificityfor the same or cross-reacting epitopes, i.e., immunospecific for thesame epitope, on ICAM-1 as the above preferred anti-ICAM-1 antibodies,or derived from the above antibodies. Thus, the present inventioncontemplates a monoclonal antibody, and immunoreactive fragmentsthereof, that has the immunospecificity of a monoclonal antibodyproduced by a hybridoma selected from the group consisting of 14E11,16G8, 2E12, 2B12, 1G12 and 2D5.

Immunological techniques for determining the immunospecificity of amonoclonal antibody are well known in the art, and can includecompetition binding studies and other cross-reaction assays. See, forexample the immunoassays described in Antibodies: A Laboratory Manual,Harlow et al., Cold Spring Harbor Laboratory, 1988.

Also contemplated by this invention is the hybridoma cell, and culturescontaining a hybridoma cell that produce a monoclonal antibody of thisinvention.

Hybridomas 14E11, 16G8, 2E12, and 2B12 have been deposited pursuant toBudapest Treaty requirements with the American Type Culture Collection(ATCC), Rockville, Md., on Jun. 10, 1992, and were assigned accessionnumbers HB 11064, HB 11063, HB 11062 and HB 11061, respectively.

Hybridomas 14E11, 16G8, 2E12, and 2B12 were deposited in a depositoryaffording permanence of the deposit and ready accessibility thereto bythe public upon the issuance of a patent, under conditions which assurethat access to the hybridomas will be available during the pending ofthe patent application to those deemed by the Commissioner to beentitled to such access, and that all restrictions on the availabilityto the public of the hybridomas as deposited will be irrevocably removedupon the granting of the patent. The deposited hybridomas will bemaintained by the ATCC for the term of the patent or 30 years from thedate of deposit, whichever is longer, and in all events for at leastfive years after the date of the last request for access.

E. Methods of Inhibiting Fibrinogen Binding To Endothelial Cells andInhibiting Fibrinogen/Endothelial Cell-Mediated Inflammation

The present invention contemplates a method of inhibiting fibrinogen(Fg) binding to endothelial cells by contacting said endothelial cellswith a composition containing a Fg or ECR homolog, or both, of thisinvention dispersed in a physiologically acceptable excipient. Themethod can be practiced both in vitro and in vivo.

The method requires that an amount of Fg or ECR homolog be used in thecontacting as to be effective at inhibiting Fg binding to theendothelial cells, i.e., an Fg-binding inhibiting amount.

As described herein, the use of a Fg homolog or an ECR homolog, or both,exhibit(s) inhibition of Fg binding to endothelial cells because thesetwo reagents mimic, as homologs, their natural counterparts and therebyblock the fibrinogen-ECR interaction as identified by the presentinvention.

In the examples herein, the ECR homolog monoclonal antibody anti-ICAM-1is as an exemplary reagent for use in a composition for the presentmethod. In another example, the Fg homolog, a polypeptide derived fromfibrinogen, is used as an exemplary reagent. However, it should beunderstood that the invention contemplates the use of any Fg or ECRhomolog and is not limited to those specific reagents.

In the method, a physiologically acceptable composition containing a Fghomolog or ECR homolog is contacted with endothelial cells in an aFg-binding inhibiting amount. Typically the amount is an amountsufficient to contact the cells with a concentration of homolog in therange of about 0.1 to 100 microgram per milliliter (ug/ml).

Insofar as the binding of Fg to endothelial cells mediates fibrinogenand endothelial cell-mediated inflammation, inhibiting Fg binding invivo provides a therapeutic method for inhibiting inflammation in apatient suffering from, or at risk for, fibrinogen and endothelialcell-mediated inflammation.

Thus, the present invention also contemplates a method of inhibitingfibrinogen/endothelial cell-mediated inflammation in a patientcomprising administering to the patient a therapeutically effectiveamount of a pharmaceutically acceptable composition comprising asubstantially pure homolog selected from the group consisting of a Fghomolog and an ICAM-1 homolog dispersed together with a pharmaceuticallyacceptable excipient (carrier).

Patients in which the inhibition of Fg binding to endothelial cells, andthe inhibition of inflammation, would be clinically useful includepatients with various types of inflammation, or at risk of inflammation,including but not limited to patients with very recent myocardialinfarction (within 40 hours of the acute event) where the Fg or ECRhomolog would prevent neutrophil accumulation on exposed tissues due toinjury to those tissues, patients with autoimmune responses, generalinflammatory or localized inflammatory reactions, glomerular nephritis,delayed type hypersensitivity, psoriasis, autoimmune thyroiditis,multiple sclerosis, rheumatoid arthritis, lupus erythematosis, tissuetransplants, graft rejection, reperfusion injury of tissue, and the likeinflammatory disorders.

The inhibition of fibrinogen/endothelial cell-mediated inflammation canbe detected by measuring changes in the amount of neutrophilaccumulation at the site of an inflammation producing injury or wound.For example, the number of neutrophils that accumulate at the site of asponge placed under the skin can be determined both before and after aFg or ECR homolog is administered to the patient. See, for example,Price et al., J. Immunol., 139:4174-4177 (1987).

Fibrinogen/endothelial cell-mediated inflammation includes any of thevarious biological processes mediated lymphocyte having a Mac-1 receptoron its cell surface. Typical biological processes include adhesion ofMac-1 bearing cells to vascular endothelium and specific interactionswith extracellular matrix proteins.

In a related embodiment, the invention contemplates methods forinhibiting tumor cell growth, particularly tumor cell adhesion and tumorcell metastasis. This embodiment is based on the established role ofendothelial cell ICAM-1 receptor in tumor cell growth and particularlymetastasis.

The mechanism of action for inhibition of tumor cell growth andmetastasis is based on the inhibition of ability of ICAM-1 to interactwith fibrinogen. Such inhibition is conducted as described herein usinga Fg or ECR homolog of this invention.

Thus, the present invention also contemplates a method of inhibitingtumor metastasis formation in a patient with tumors comprisingadministering to the patient a therapeutically effective amount of apharmaceutically acceptable composition comprising a Fg or ECR homologof this invention. Particularly preferred are the Fg homologpolypeptides described herein. Dosages and routes of administration aresubstantially the same as for methods for inhibiting Fg binding toendothelial cells.

A homolog is typically administered according to the present inventionas a pharmaceutically acceptable composition in the form of a solutionor suspension. However, as is well known, peptides and proteins such asa Fg or ECR homolog can also be formulated for therapeuticadministration as tablets, pills, capsules, sustained releaseformulations or powders. Typically, suitable dosage ranges for antherapeutic composition are of the order of one to hundreds of nanomolesof Fg or ECR homolog per kilogram body weight per minute and depend onthe route of administration. In any case, the administered compositioncontains at least about 0.10% to about 99% by weight of a Fg or ECRhomolog per weight of composition, preferably 10%-90% and morepreferably 25-75%.

The composition is administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's blood hemostatic system to utilize the active ingredient, anddegree of inflammation inhibition or fibrinogen binding inhibitiondesired. Precise amounts of active ingredient required to beadministered depend on the judgement of the practitioner and arepeculiar to each individual.

A therapeutically effective amount of homolog can be expressed as anamount sufficient to produce a final concentration of homolog in apatient's blood. That blood concentration can be determined by an invitro assay for the homolog in a liquid body sample (e.g., blood), suchas is described herein, or can be calculated based on the patient's bodyweight and blood volume as is well known.

Suitable dosage ranges of a homolog for the therapeutic methodsdescribed herein are in the order of about 0.1 to about 20 milligrams,preferably one to ten milligrams of homolog per kilogram of body weightof patient per day, and depending on the route of administration. Stateddifferently, a therapeutically effective dosage is an amount sufficientto produce an intravascular concentration of in the blood of the patientin the range of about 0.1 to about 100 micrograms/milliliter (μg/ml),preferably about 10 to about 20 μg/ml of the active ingredient.

F. Methods of Detecting Homolog

The present invention contemplates any method that results in detectinga homolog by producing a reaction product using a monoclonal antibody,polyclonal antibody, or homolog binding reagent.

Due to the binding interaction of a Fg homolog and an ECR homolog, a Fghomolog binding reagent can be any ECR homolog, and an ECR homologbinding reagent can be any Fg homolog.

Those skilled in the art will understand that there are numerous wellknown clinical diagnostic chemistry procedures that can be utilized toform and detect such reaction products. Thus, while exemplary assaymethods are described herein, the invention is not so limited.

Various heterogeneous and homogeneous assay protocols can be employed,either competitive or non-competitive for detecting the presence andpreferably the amount of a Fg or ECR homolog in a tissue or liquidcomposition.

A Fg or ECR homolog may be detected in any sample such as a solid,liquid or body fluid sample. In preferred embodiments a homolog isdetected in body fluid samples include blood, plasma, serum, mucous,sputum and the like.

A homolog may also be detected in vitro or in vivo in various tissuesand organs. In preferred embodiments tissue slices or tissue sectionsmay be assayed for the presence and location of a homolog. In otherpreferred embodiments organs may be assayed in vivo for the presence andto determine the location of a homolog.

Detection of the amount of homolog present in vitro or in vivo is usefulbecause the amount of homolog present correlates with the progress oftherapeutically administered homolog present in the patient beinganalyzed. Thus determination of the amount of homolog present in thepatient being analyzed allows the therapeutic administration of ahomolog to a patient to be monitored to determine the clinical state ofthe patient.

In one embodiment the present invention contemplates a method ofdetecting the presence and preferably the amount, of a Fg homolog in aliquid composition. The steps of this method include:

(1) admixing a sample of endothelial cells with a predetermined amountof a liquid sample containing a Fg homolog and a predetermined amount oflabelled Fg homolog to form a competition reaction admixture;

(2) maintaining the reaction admixture formed in step (1) for apredetermined time period sufficient for the Fg homolog present in theliquid composition to bind to the endothelial cells and form aendothelial cell:Fg homolog complex and to allow the labelled Fg homologto bind the endothelial cells and form a labelled endothelial cell:Fghomolog complex;

(3) assaying for the presence and/or amount of labelled endothelialcell:Fg homolog complex formed in step (2) thereby detecting thepresence and/or amount of a Fg homolog in the composition.

A predetermined amount of a liquid composition containing a Fg homologis a known amount of Fg-containing liquid composition that is useful andeasily assayed. This predetermined amount of Fg homolog containingliquid composition has been shown to be useful by performing a series oftest assays with an amount of liquid composition containing a knownconcentration of Fg homolog and is sufficient to allow the assay to beperformed. Preferred amounts of a liquid composition are from about 1microliter (μl) to about 1000 μl.

The liquid composition also contains a labelled Fg homolog. A label isan atom or molecule that is either directly or indirectly involved inthe production of a detectable signal to indicate the presence of acomplex. Any label or indicating means can be linked to or incorporatedin an expressed protein, polypeptide, or antibody or monoclonal antibodycomposition of the present invention, or used separately, and thoseatoms or molecules can be used alone or in conjunction with additionalreagents. Labels include various in vivo labels useful within the bodyof a patient such as ¹¹¹In, ⁹⁹Tc, ⁶⁷Ga, ¹⁸⁶Re, and ¹³²I.

The label can be a fluorescent labelling agent that chemically binds toantibodies or antigens without denaturing them to form a fluorescent(dye) that is a useful immunofluorescent tracer. Suitable fluorescentlabelling agents are fluorochromes such as fluorescein isocyanate (FIC),fluorescein isothiocyanate (FITC), 5-dimenthylamin-1-naphthalenesulfonylchloride (DANSC), tetramethylrhodamine isothiocyanate (TRITC),lissamine, rhodamine 8200 sulphonyl chloride (RB 200 SC) and the like. Adescription of immunofluorescence analysis techniques in found inDeluca, “Immunofluorescence Analysis”, in Antibody As A Tool,Marchalonis et al., eds., John Wiley & Sons, Ltd., pp. 189-231 (1982),which is incorporated herein by reference.

In preferred embodiments the label is an enzyme, such as horseradishperoxidase (HRP), glucose oxidase, or the like. In such cases where theprincipal indicating group is an enzyme such as HRP or glucose oxidase,additional reagents are required to visualize the fact that areceptor-ligand complex (immunoreactant) has formed. Such additionalreagents for HRP include hydrogen peroxide and an oxidation dyeprecursor such as diaminobenzidine. An additional reagent useful withglucose oxidase is 2,2′-azino-di (3-ethyl-4-2-benzthiazoline-G-sulfonicacid) (ABTS).

Radioactive elements are also useful as labels. An exemplary radiolabelis a radioactive element that produces gamma ray emissions. Elementswhich themselves emit gamma rays, such as ¹²⁴I, ¹²⁵I, ¹²⁸I, ¹³¹I, ¹³²I,and ⁵¹Cr represent one class of gamma ray emission-producing radioactiveelement indicating groups. Particularly preferred is ¹²⁵I. Another groupof useful indicating groups are those elements such as ¹¹C, ¹⁸F, ¹⁵O and¹³N which themselves emit positrons. The positrons so emitted producegamma rays upon encounters with electrons present in the animal's body.Also useful is a beta emitter, such as indium.

The linking of labels, i.e., labelling of, polypeptides and proteinssuch as a Fg homolog is well known in the art. For instance, antibodymolecules produced by a hybridoma can be labelled by metabolicincorporation of radioisotope-containing amino acids provided as acomponent in the culture medium. See, for example, Galfre et al., Meth.Enzymol., 73:3-46 (1981). The techniques of protein conjugation orcoupling through activated functional groups are particularlyapplicable. See, for example, Aurameas et al., Scand. J. Immunol., Vol.8, 7:7-23 (1978), Rodwell et al., Biotech., 3:889-894 (1985), and U.S.Pat. No. 4,493,795, which is incorporated herein by reference. Inaddition, site directed coupling reactions can be carried out so thatthe label does not substantially interfere with the ability of theantibody molecules to bind their specific antigen. See, for example,Rodwell et al., Biotech., 3:889-894 (1985).

Alternatively, where polypeptides are used as a homolog, a label can beincorporated by adding amino acid residues to either termini of thepolypeptide adapted for labelling. For example, iodinated tyrosine canbe used by the addition of a tyrosine residue to the polypeptide. Aexemplary labelled polypeptide has the tripeptide KYG added to the aminoterminus, with the tyrosine iodinated, using the polypeptide shown inSEQ ID NO 4.

The reaction admixture is maintained for a predetermined time periodsufficient for the Fg homolog and the labelled Fg homolog present in theliquid composition to bind to the endothelial cells and form anendothelial cell:Fg homolog complex and a labelled endothelial cell:Fghomolog complex.

The amount of time sufficient for the Fg homolog and the labelled Fghomolog to bind the endothelial cells depends upon several physicalparameters including temperature and the concentration of the variousreactants. In preferred embodiments, the predetermined time period isfrom about 1 minute to 24 hours. In more preferred embodiments thepredetermined time period is from about 10 minutes to about 1 hour. Inthe most preferred embodiments, the predetermined time period is fromabout 15 minutes to 30 minutes. Typically this time period ispredetermined to optimize the assay.

Typically the reaction admixture is maintained under biological assayconditions that maintain the activity of the polypeptide and proteinmolecules including the Fg homolog and the endothelial cell sought to beassayed, and include a temperature range of about 4 degrees C. (4° C.)to about 45° C., a Ph value range of about 5 to about 9 and an ionicstrength varying from that of distilled water to that of about one molarsodium chloride. Methods for optimizing such conditions are well know inthe art.

The presence of labelled endothelial cell:Fg homolog complex formed bymaintaining the reaction admixture in step (2) is assayed.

The direct or indirect methods used to assay for the presence of andpreferably the amount of labelled endothelial cell:Fg homolog complexformed depend on the particular label used and are well known in theart. For example, the amount of radioactivity in the labelledendothelial cell:Fg homolog complex may be determined as described inExample 5. Alternatively, homogeneous assay methods such as thosedescribed in U.S. Pat. No. 4,536,479; No. 4,233,401; No. 4,233,402 andNo. 3,996,345, whose disclosures are incorporated herein by reference.

In other preferred embodiments, the present invention contemplatesanother method of detecting the amount of a Fg homolog in a liquidsample using an Fg homolog binding reagent, ie, an ECR homolog. Thesteps of this method include;

(1) admixing an ECR homolog with a predetermined amount of a liquidsample containing a Fg homolog to form an binding reaction admixture;

(2) maintaining the binding reaction admixture formed in step (1) for apreselected time period sufficient for the Fg homolog present in theliquid sample to bind to the ECR homolog and form a complex containingFg homolog and ECR homolog; and

(3) determining the amount of the complex formed in step (2), therebydetecting the amount of a Fg homolog within the liquid sample.

A preferred ECR homolog is an anti-Fg monoclonal antibody, and thecomplex formed is an immunoreaction complex.

In a related embodiment, the invention contemplates a method for thedetection of the amount of an ECR homolog in a liquid sample using anECR homolog binding reagent, ie, a Fg homolog. The method is practicedin the same manner as above, except that an Fg homolog is added as thebinding reagent to a sample containing an ECR homolog.

Preferably, the liquid sample containing a homolog is a biological fluidsample such as blood, plasma, serum, sputum, saliva, and the like.Preferably, the amount of liquid sample admixed is known.

For the determining step, it is preferred that the added homolog (Fghomolog for detecting ECR homolog, and ECR homolog for detecting Fghomolog) is labelled, i.e., operatively linked to an indicating meanssuch as an enzyme, radionuclide and the like as described earlier. Inthis embodiment, the determination is made by detecting thepresence/amount of the label in the complex, thereby determining thepresence/amount of the homolog in the sample.

In one embodiment, the added homolog is present as part of the solidsupport, i.e., operatively linked to a solid matrix, so that thereaction admixture formed is a solid and liquid phase, with theobjective of “capturing” the sample to be determined.

The reaction admixture is maintained for a predetermined time periodsufficient for the homolog present in the liquid sample to bind to theantibody and form a complex containing a homolog to be detected and theadded homolog.

Biological assay conditions are those conditions that maintain thebiological activity of the reagents and the homolog to be assayed asdiscussed earlier.

In a preferred embodiment, the amount of homolog in the complex can bedetermined, either directly or indirectly, using assay techniques wellknown in the art, and typically depend upon the type of indicating meansused.

G. Detection of ECR Receptors in vivo

A method of detecting the presence and preferably the amount andlocation of cells having ECR receptors in a mammal is contemplated. Aneffective amount of a composition containing a physiologically tolerablediluent and an amount of Fg homolog linked to an in vivo indicatingmeans is parenterally administered to a human subject. Parenteraladministration includes intramuscular administration, intravenousadministration, and administration into other body sites, such assynovial fluid. The amount of composition administered is sufficient tobind a detectable quantity of ECR receptors. In preferred embodimentsthe Fg homolog is anti-ICAM-1 antibody molecules, or D₃₀ fragment.

As used herein, the terms “label” and “indicating means” in theirvarious grammatical forms refer to single atoms and molecules that areeither directly or indirectly involved in the production of a detectablesignal to indicate the presence of a complex. “In vivo” labels orindicating means are those useful within the body of a human subject.Any label or indicating means can be linked to or incorporated in anexpressed protein, polypeptide, or antibody molecule that is part of anantibody or monoclonal antibody composition of the present invention, orused separately, and those atoms or molecules can be used alone or inconjunction with additional reagents. Such labels are themselves wellknown in clinical diagnostic chemistry and constitute a part of thisinvention only insofar as they are utilized with otherwise novelproteins methods and/or systems.

The linking labels, i.e., labelling of, polypeptides and proteins iswell known in the art. For instance, antibody molecules produced by ahybridoma can be labelled by metabolic incorporation ofradioisotope-containing amino acids provided as a component in theculture medium. See, for example, Galfre et al., Meth. Enzymol., 73:3-46(1981). The techniques of protein conjugation or coupling throughactivated functional groups are particularly applicable. See, forexample, Aurameas et al., Scand. J. Immunol., Vol. 8, 7:7-23 (1978),Rodwell et al., Biotech., 3:889-894 (1984) and U.S. Pat. No. 4,493,795.

The subject is then maintained for a predetermined time periodsufficient for the Fg homolog to bind to the ECR receptors present onthe cells of the human subject and form a ECR:Fg homolog complex.Preferably, this time period has been predetermined to optimize theformation of an ECR:Fg homolog complex.

The subject is then assayed for the presence of and preferably thelocation of any ECR:Fg homolog complexes formed.

H. Method For Identifying Inhibitors

The present invention also contemplates methods for identifying acomposition that inhibits the fibrinogen binding interaction toendothelial cells where the interaction is mediated by the fibrinogenbinding site on ECR as described herein.

The method is generally useful for the design of novel therapeutics usedin the inhibition of endothelial cell/fibrinogen mediated inflammation,and is particularly useful as a mass screening procedure to identifyactive inhibitor compounds and formulations.

The invention therefore contemplates a method for identifying acomposition which inhibits fibrinogen binding to ECR on endothelialcells, which comprises:

(a) incubating components comprising the composition to be testedtogether with an ECR homolog and a Fg homolog under conditions whichallow the ECR homolog to interact and bind with the Fg homolog; and

(b) measuring the interaction of the ECR homolog with the Fg homolog,thereby measuring the capacity of the composition to inhibit theinteraction.

A preferred ECR homolog is ICAM-1, and a preferred Fg homolog is Fg, asdefined herein.

The measuring can be directed at detecting free Fg homolog, free ECRhomolog, or free composition. Alternatively, the measuring can detectthe binding interaction of the composition with either the ECR homologor the Fg homolog. Typically, the binding interaction is measured bydetecting a complex formed upon binding.

Conditions sufficient for a binding interaction are generallyphysiological, and are time temperature and buffer conditions compatiblewith the binding of fibrinogen onto endothelial cells, as shown in theExamples.

More preferably, the binding interaction is detected in assays where oneor the other of Fg homolog and ECR homolog are in the solid phase, andthe other is labelled. The measuring comprised detecting the presence,and preferably amount of label in the solid phase, directly indicatingthe amount of inhibition by the composition.

In a related embodiment, the invention describes a method of screeningfor compositions effective at inhibiting fibrinogen binding to ECRcomprising the steps of:

a) admixing in an inhibition reaction admixture preselected amounts of aputative inhibitor composition, a fibrinogen homolog, and an ECR homologas defined herein;

b) maintaining said admixture under conditions sufficient for said ECRhomolog to bind to said Fg homolog and form an ECR homolog:Fg homologcomplex; and

c) measuring the amount of ECR homolog:Fg homolog complex formed in step(b), and thereby the effectiveness of said inhibitor composition.

In practicing the method, one of the homologs is labelled and in theliquid phase, and the other homolog is in the solid phase, wherein themeasuring involves detecting the amount of label in the solid phase.Other formats are readily apparent.

Preferably, the ECR homolog is purified ICAM-1. More preferably, the ECRhomolog is in the solid phase. Still more preferably, the solid phase isa cell on which ECR is located such as an endothelial cell, lymphoidcell, or a recombinant cell capable of expressing recombinant ICAM-1.

Exemplary screening methods are described in Example 5 where antibodieswhere identified that inhibit Fg binding to endothelial cells. Fghomologs and ECR homologs can also be developed and/or identified by theabove methods.

EXAMPLES

The following examples are intended to illustrate, but not limit, thepresent invention.

1. Preparation of Fibrinogen Analogs

A. Purification of Plasma Fibrinogen

Fibrinogen was isolated from fresh plasma by cold ethanol fractionationprocedures. To one volume of plasma, 0.22 volumes of cold 50% ethanol,pH 7.0 was admixed which lowered the temperature to −3 degrees Celsius(−3° C.). The admixture was centrifuged and the resultant precipitatewas washed with 0.5 original volumes (OV) of 7% ethanol, pH 6.5 at −3°C. The precipitate was re-collected and dissolved in 0.25 OV of 0.55 Mtrisodium citrate buffer, pH 6.5 at 30° C. The resultant solution wascooled to 0° C. and the fibrinogen was precipitated by the addition ofcold 20% ethanol to a final concentration of 8% to form purifiedfibrinogen.

To remove any possible contamination of the purified fibrinogen withfibronectin, the purified fibrinogen preparation was passed over agelatin Sepharose™ 4B column (Pharmacia LKB, Piscataway, N.J.) accordingto manufacturer's instructions resulting in fibronectin-free fibrinogen.

B. Preparation of D₃₀ from Purified Fibrinogen

1) Proteolytic Digestion of Purified Fibrinogen

Fifty milligrams (mg) of purified fibrinogen prepared in Example 1A wasdissolved in 1 milliliter (ml) of a TBS buffer solution containing 0.01M Tris(hydroxymethyl)aminomethane (Tris-HCl), 0.14 M sodium chloride(NaCl), pH 7.4, and was proteolytically digested byStreptokinase-activated plasminogen (plasmin) according to the followingprocedure.

Streptokinase-activated plasminogen was prepared by admixing plasminogen(KABI, 20 units (U)) to 2 ml of 0.1 M sodium phosphate buffer, pH 7.4and pre-maintaining for 10 minutes at 37° C. with 500 U of streptokinase(Streptase, Behring). This solution was then admixed at a finalconcentration of 18 micrograms per ml (μg/ml) to the solution ofpurified fibrinogen in 2 M urea.

The admixture was maintained for 2 hours at 37° C. The proteolyticreaction in the admixture was terminated by the addition of 50,000 U/mltrasylol (Sigma Chemical Co., St. Louis, Mo.). The resulting solution offibrinogen fragments was extensively dialyzed against a solution of TBSfor 24 hours at 4° C. The dialysis buffer was changed every 8 hours. Thedialyzed solution was then recovered and applied on a Sephadex™ G-100column (Pharmacia LKB). The column chromatography was performed toseparate the fragments resulting from the proteolytic digestion offibrinogen. The column was prewashed with a running buffer of TBSfollowed by application of the dialyzed fibrinogen fragments. Fractionsof 3 ml were collected and the molecular weights of the separatedfragments in the fractions were determined by 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with and withoutreduction by 3% mercaptoethanol.

Three fragments of different molecular weights were visualized byCoomassie Blue staining of the gel. Fragments X, D, and E had respectivemolecular weights of approximately 240,000, 85,000, and 50,000 undernon-reducing conditions. The fractions corresponding to the threeseparate peaks were separately pooled, dialyzed against distilled water,and concentrated by lyophilization.

2) Proteolytic Digestion of Fragment D to Produce a D₃₀ Homolog

Fibrinogen fragment D with a molecular weight of 80,000 (80 kilodaltons(kD)), purified and concentrated, was proteolytically digested withplasmin in 2 M urea for 24 hours at 37° C. as described in Example 1B.The digestion was terminated and the resultant solution dialyzed asdescribed in Example 1B. The dialyzed solution was recovered and theproducts of the digestion were isolated by high performance liquidchromatography (HPLC) on a Mono-Q-column (Pharmacia LKB) equilibrated in0.01 M sodium phosphate, pH 7.0. The fragments were eluted with asolution of 0.01 M sodium phosphate and 1 M sodium chloride, pH 7.0. Thepurity of the eluted proteins in the collected fractions was assayed on15% SDS-PAGE under non-reducing conditions. Coomassie Blue staining ofthe gel revealed a 30 kD fragment of greater than 90% homogeneity. Thepurified proteolytic digestion product of fragment D having a kD of 30was designated D₃₀. The peak fractions containing D₃₀ were pooled andconcentrated by lyophilization.

2. Purification of the Endothelial Cell RGD-Independent FibrinogenReceptor

A. Preparation of an Human Umbilical Vein Endothelial Cell Lysate

Human umbilical vein endothelial cells, (HUVEC) commercially availablefrom Clonetics, San Diego, Calif., were passaged into 40 gelatin-coatedT75 tissue culture flasks (Falcon, Thousand Oaks, Calif.) and maintainedin endotoxin-free RPMI 1640 (Whittaker M. A. Bioproducts, Walkersville,Md.) supplemented with 10% fetal bovine serum (FBS) (Hyclone, SterileSystems, Logan, Utah), 25 mM Hepes[4-(2-hydroxyethyl)-1-piperidineethanesulfonic acid] (CalbiochemBoehring, La Jolla, Calif.), 100 μg/ml penicillin-streptomycin-fungizone(Whittaker), 0.5% endothelial cell growth factor (BiomedicalTechnologies, Stoughton, Mass.) and 1 mM glutamine (Whittaker). In orderto increase the yield of purified endothelial cell receptor (ECR), thecultured endothelial cells, at a density of approximately 5×10⁶cells/flask, were stimulated 6 hours prior to harvesting by exposure totumor necrosis factor alpha (TNF, Genzyme Corp., Cambridge, Mass.) at aconcentration of 20 ng/ml. The cellular response to TNF was initiallyrevealed in experiments described in Example 3A. After the culturemedium was removed, the cells were detached from the flasks with 4 mMethylenediaminetetraacetic acid (EDTA, Sigma Chemical Co.) at 37° C. for30 minutes. The detached cell suspensions from all the flasks werepooled and pelleted by centrifugation at 1200 rpm for 10 minutes. Thepelleted cells were washed twice with cold phosphate-buffered saline(PBS).

After the final wash, the cells were resuspended in PBS containing 1millimolar (mM) calcium chloride (CaCl₂) and 1 mM magnesium chloride(MgCl₂) in preparation of labelling cell surface proteins with 4 mCi ofsodium ¹²⁵iodide (¹²⁵I) (NEN Du Pont de Nemours, Wilmington, Del.) bythe lactoperoxidase iodination method known to one skilled in the artand as described in “Antibodies: A Laboratory Method”, Eds Harlow etal., Cold Spring Harbor Laboratory, pp 434-435 (1988). After thelabelling procedure, the cells were washed 3 times with PBS lacking allcations. After the final centrifugation wash, the pellet was frozen,thawed then resuspended in a volume of 3 parts Tris-buffered salineextraction buffer (TBS extraction buffer) to 1 part pellet. TBSextraction buffer consisted of 25 mM Tris-HCl, 136 mM NaCl and 2 mMpotassium chloride (KCl) that also contained 1-2 mM MgCl₂ , 1-2 mMmanganese chloride (MnCl₂), 50 mM octyl beta glucopyranoside, 1 mMphenylmethylsulfonyl-fluoride (PMSF), 1 μg/ml aprotinin, 1 μg/mlleupeptin, 1 μg/ml pepstatin and 1 μg/ml alpha 2 macroglobulin. Thepreferred concentrations of MgCl₂ and MnCl₂ were 1 mM. Calcium chloridewas absent from the extraction buffer and all subsequent buffers used inthe isolation and Purification of the ECR.

The resultant cell lysate having 3 ml was centrifuged at 3000×g topellet the insoluble cellular debris. The supernatant containing theisolated ECR was removed and labelling efficiency was determined bygamma detection. Approximately a specific activity of 200,000 counts perminute (cpm) per 10 microliters (μl) was obtained by the labellingprocedure.

B. Purification of Labelled ECR by Affinity Chromatography on aFibrinogen Sepharose™ Column

1) Sequential Elutions of Fibrinogen Affinity Column

The labelled cell supernatant prepared above was precleared prior topurification by chromatography over a plain Sepharose™ CL4B column(Pharmacia) previously equilibrated with the TBS extraction buffer. Forpurification of the ECR by affinity chromatography, the flow-throughcontaining the labelled ECR was collected from the plain Sepharose™column and loaded onto a fibrinogen Sepharose™ column prewashed with 10column volumes of TBS extraction buffer. The column was previouslyprepared by coupling 8 mg of purified fibrinogen prepared in Example 1Ato one ml (approximately 0.333 grams of resin) of cyanogenbromide-activated (CNBr) Sepharose™ 4B according to manufacturer'sinstructions (Pharmacia LKB). The labelled ECR-containing solution wasmaintained on the fibrinogen column overnight at 4° C. and was mixedoccasionally to immobilize the ECR on the fibrinogen ligand. Followingthe maintenance period, the flow-through was collected and storedseparately at 4° C.

The column was then washed with 10 column volumes of TBS extractionbuffer. Prior to the EDTA elution of the ECR immobilized on thefibrinogen column, the column was first maintained with 300 μl of a 1mg/ml Arg-Gly-Glu (RGE) peptide solution dissolved in TBS extractionbuffer to elute nonspecifically immobilized labelled proteins. Thecollection of the eluate was followed by a 10 minute waiting periodbefore the next application of 300 μl of the RGE solution to the column.The elution with RGE was repeated 10 times for a total of 10 collectedfractions. For the second set of elutions to remove contaminatingfibrinogen-bound labelled vitronectin receptor, a member of the integrinsuperfamily, the column was maintained with 300 μl of a 1 mg/mlArg-Gly-Asp (RGD) peptide solution dissolved in TBS extraction buffer.The RGD eluate was collected and the elution protocol was repeated 10separate times as described for the RGE elution resulting in thecollection of labelled vitronectin receptor over 10 fractions. Peakfractions were determined by gamma detection. The peptides used for theabove-described elutions were synthesized using the classicalsolid-phase technique described by Merrifield, Adv. Enzymol., 32:221-296(1969) as adapted for use with a model 430 automated peptide synthesizer(Applied Biosystems, Foster City, Calif.). Prepared polypeptide resinswere cleaved by hydrogen fluoride, extracted and analyzed for purity byhigh-performance liquid chromatography (HPLC) using a reverse-phase C18column manufactured by Waters Associates, Milford, Mass.

Labelled ECR bound to the then vitronectin receptor-free fibrinogencolumn was then eluted with 10-20 mM EDTA dissolved in TBS extractionbuffer. The 20 mM EDTA elution was preferred. The elution protocol wasperformed as described above resulting in the collection of purified ECRover 10 separate fractions. Peak fractions containing the eluted¹²⁵I-labelled ECR were determined by gamma detection. The column wasthen washed with 10 column volumes of TBS extraction buffer followed by3 column volumes of 1 M NaCl in TBS. The column was stored at 4° C.after a final wash with at least 20 column volumes of PBS containing0.02% sodium azide.

2) Characterization of the Purified Fibrinogen-Specific ECR

The molecular weight of the fibrinogen Sepharose™-purified ¹²⁵I-labelledECR was determined by 7.5% SDS-PAGE with and without reduction with 3%beta-mercaptoethanol. FIG. 1 shows the autoradiographic results ofelectrophoresis of aliquots of peak fractions from both the RGD and EDTAelutions of cell lysates prepared from cells either left untreated ortreated with TNF as described in Example 2A. Lanes 1 and 3 show theRGD-eluted receptors isolated from cell lysates respectively preparedfrom untreated or TNF-treated cells. No bands are detectable in lane 1.However, in lane 3, two bands corresponding to the approximate molecularweights of 125 and 110 kD are present. These bands respectivelycorrespond to alpha v and beta 3 subunits of the vitronectin receptor asdescribed by Cheresh et al., Proc. Natl. Acad. Sci., 84:6471-6475(1987), hereby incorporated by reference. Lanes 2 and 4, respectivelyEDTA-eluted ECR isolated from untreated and TNF-treated cells, revealthe presence of a lower molecular weight band of approximately 90-95 kD,the intensity of which is enhanced about 3-5 fold as a result of theinduction of ECR expression by exposure to TNF. Thus, the EDTA-elutedfractions contained a non-RGD dependent ECR having a molecular weight of90-95 kD distinct from the vitronectin receptor that binds to fibrinogenvia the RGD tripeptide sequence (Cheresh et al., supra).

C. Purification of Labelled ECR by Affinity Chromatography on a RGDSepharose™ Column Followed by a Fibrinogen Sepharose™ Column

1) Sequential Column Chromatography

That the EDTA-eluted fibrinogen-binding ECR was a receptor distinct fromvitronectin receptor was confirmed using a alternative approach ofpurifying ECR from labelled cell lysates by affinity chromatography overtwo different columns. The cell lysate prepared in Example 2A was firstapplied onto an RGD Sepharose™ column previously equilibrated with TBSextraction buffer to immobilize RGD-specific receptors to theSepharose™-bound RGD. Coupling of RGD to CNBr-Sepharose™ was performedas described in Example 2A for preparation of a fibrinogen Sepharose™column. After the overnight maintenance period for maximizing theinteraction of the ¹²⁵I-labelled cell lysate containing RGD-dependentreceptors with the Sepharose™-bound RGD, the flow-through was collectedand applied on a fibrinogen Sepharose™ column as described in Example2B. The RGD column was then washed as previously described. EDTA elutionbuffer was subsequently applied to the washed RGD column and fractionscontaining ¹²⁵I-labelled EDTA-eluted receptors were collected asdescribed for the elution protocol in Example 2B. The characterizationof the EDTA-eluted receptors from the RGD Sepharose™ column is describedin Example 2C2) below.

The flow-through from the RGD column was maintained overnight with thefibrinogen column to allow for maximum interaction of the RGD-extractedcell lysate containing RGD-independent fibrinogen receptors with theSepharose™-bound fibrinogen. After the maintenance period, the¹²⁵I-labelled receptors bound to fibrinogen were eluted following thesame procedure as described for the elution from the fibrinogen columnin Example 2B. Ten fractions were collected from each of the sequentialRGE, RGE and EDTA elutions. The characterization of the RGD-andEDTA-eluted material from the fibrinogen Sepharose™ column is describedin Example 2C2) below.

2) Characterization of the RGD—Versus Fibrinogen-Dependent Receptors

Aliquots of the collected fractions from the EDTA elution of the RGDSepharose™ column were electrophoresed adjacent to aliquots from both ofthe RGD and EDTA elutions from the fibrinogen Sepharose™ column underboth nonreducing and reducing conditions to provide for an optimalcomparison and characterization of the eluted receptors. Aliquots of thecollected fractions were electrophoresed as described in Example 2B2).The results of autoradiographic exposure of the electrophoresed¹²⁵I-labelled receptors are shown in FIG. 2 in 8 lanes. Lanes 1 through4 show migration of proteins under reducing conditions while lanes 5through 8 show the migration of identical aliquots run under nonreducingconditions. The molecular weight determinations of the electrophoresedeluted receptors are made by comparison to ¹²⁵I-labelled molecularweight standards of 210, 107, 71 and 41 kD, respectively, myosin,beta-galactosidase, bovine serum albumin and ovalbumin. These markersare shown in lanes 4 and 8.

In lanes 3 and 7, the vitronectin receptor eluted with EDTA from theRGD-Sepharose™ column exhibits the characteristic profile of alphav/beta 3 under reducing and nonreducing conditions. Unreduced alpha vhas a molecular weight of 150 kD (lane 7, upper band) which is cleavedinto two polypeptides of 125 and 25 kD under reducing conditions (lane3, middle band—the 25 kD fragment has run off the gel). Unreduced beta 3has a molecular weight of 90 kD (lane 7, lower band) which is increasedto 110 kD under reducing conditions (lane 3, lower band).

In addition to the vitronectin integrin receptor having alpha v and beta3 subunits, another integrin beta subunit, beta 1, was eluted from theRGD Sepharose™ column with EDTA. Alpha v has been shown to separatelyassociate with both beta 3 and beta 1 as described by Vogel et al., J.Biol. Chem., 265:5934-5937 (1990). Beta 1 migrates as a 120 kD proteinunder nonreducing conditions (lane 7, middle band) which increases to140 kD under reducing conditions (lane 3, upper band). Thus, theRGD-dependent vitronectin receptor consisting of alpha v/beta 3 subunitswas eluted from both a fibrinogen Sepharose™ column with RGD and from aRGD column with EDTA.

In contrast, a different profile of the eluted proteins was obtainedfrom fibrinogen affinity chromatography of cell lysates precleared onthe RGD Sepharose™ column. As described above under Example 2C1), theflow-through collected from the RGD Sepharose™ column lacking alphav/beta 1 and beta 3 was then chromatographed on a fibrinogen Sepharose™.Since all the RGD-dependent receptors were removed by the first affinitycolumn chromatography run, no ¹²⁵I-labelled elution products wereobtained when the fibrinogen column was subjected to RGD elution (lanes1 and 5, respectively, reduced and nonreduced conditions). However, withEDTA elution following the RGD elution, a single band of approximately90-95 kD under nonreducing conditions (lane 6) was recovered. Underreducing conditions, the molecular weight of the EDTA-eluted fibrinogenreceptor derived from human umbilical vein endothelial cells (HUVEC)referred to as ECR only slightly increased (lane 2). The determinedmolecular weights of the isolated ECR purified by either of the twoapproaches described in Examples 2B (sequential elutions of a singlefibrinogen Sepharose™ affinity chromatography) or 2C (sequentialaffinity chromatography on separate affinity columns) were the same.Thus, the identical ECR was purified using two alternative approaches asshown in both FIGS. 1 and 2. In addition, the ECR was also purified byaffinity chromatography on a fibrinogen Sepharose™ column with EDTAwithout a prior elution step with RGD to remove other fibrinogen-bindingreceptors.

D. Identification of the Purified Fibrinogen-Specific ECR as ICAM-1 byImmunoprecipitation

1) Immunoprecipitation of a 90-95 kD ECR with Anti-ICAM-1 MonoclonalAntibodies

Aliquots of fractions containing ¹²⁵I-labelled 90-95 kD ECR purified byeither approach as described in Example 2B or 2C were used inimmunoprecipitations to further identify the fibrinogen-binding ECR.Fifty to 100 μl of peak fractions containing the ECR as determined byaffinity chromatography as described above were separately admixed with20 μg of a mouse monoclonal anti-human Intercellular Adhesion Molecule-1(ICAM-1) antibody commercially available from Becton DickinsonImmunocytochemistry Systems, Mountain View, Calif. Separate aliquotswere admixed with control antibodies. For the IgG control, a mousemonoclonal designated 1C10 commercially available from Telios, SanDiego, Calif., was used as a control which recognized a 130 kDendothelial cell surface protein. For the IgM control, an irrelevant IgMmouse monoclonal antibody was used. The admixtures were maintained onice for one hour to form immune complexes. To immunoprecipitate orcollect the formed immune complexes, 100 μl of goat anti-mouse IgGcoupled to agarose (Sigma Chemical Co.) at a ratio of 1:1.

Typically, 50 μl of the eluted ECR in a peak fraction was admixed with50 μl of the goat anti-mouse IgG-coupled agarose and maintained on icefor 30 minutes. The immune complexes bound to goat anti-mouse agarosewere subsequently pelleted by centrifugation at 10,000×g for one minuteat 4° C. The resultant supernatants were removed by aspiration and thepellets were resuspended in TBS extraction buffer. The pellets werewashed 3 times and finally resuspended in Laemmli sample buffer forSDS-PAGE analysis against the molecular weight standards describedabove. Following electrophoresis, the gel was dried andautoradiographed. A single 90-95 kD band was evident on the developedfilms indicating that the 90-95 kD fibrinogen affinity purified ECR wasin fact ICAM-1 as determined by immunoprecipitation with a mousemonoclonal antibody raised against human ICAM-1.

2) Immunoprecipitation of a 90-95 kD ECR with Anti-HUVEC MonoclonalAntibodies

Immunoprecipitations as described above were also performed with mousemonoclonal antibodies raised against intact unstimulated HUVEC. Thepreparation and characterization of four such monoclonal antibodies isdescribed in Example 4. Fifty μl of the IgM monoclonal antibodydesignated 2E12 was admixed with 50 μl of the same fraction used in theimmunoprecipitations with the commercially available anti-ICAM-1antibody. After electrophoresis and exposure of the autoradiographicfilm, a 90-95 kD band was evident. Thus, the monoclonal antibodiesdirected against HUVEC cell surface proteins immunoprecipitated the same90-95 kD protein as that immunoprecipitated with a commerciallyavailable mouse monoclonal antibody to human ICAM-1. The ECR that bindsto fibrinogen via a RGD-independent binding site is now identified asICAM-1 as determined by affinity chromatography analysis (Examples 2Band 2C). The binding of the ECR, hereinafter referred to as ICAM-1, toan RGD-independent binding site in fibrinogen is a novel finding.

3. Confirmation of an RGD-Independent Fibrinogen Receptor on EndothelialCells (HUVEC)

Adhesion of leukocytes to vascular endothelium is one of the earliestevents in a variety of immune-inflammatory reactions. At the molecularlevel, leukocyte adhesion to endothelial cells is a redundant mechanism,supported by the regulated recognition of a disparate set of membranereceptors expressed on both leukocytes and endothelial cells, the latterof which may either be in a resting or a cytokine-stimulated state. Anovel set of molecular interactions participating in leukocyte adhesionare now identified. Fibrinogen has been shown to interact leukocytes(monocytes, peripheral mononuclear cells and various cell lines) via theintegrin CD11b/CD18, also referred to as Mac-1, as described by Altieriet al., J. Biol. Chem., 265:12119-12122 (1990), hereby incorporated byreference. Studies of the interaction of fibrinogen with endothelialcells in vitro has now resulted in the discovery of an endothelial cellsurface membrane receptor that binds an RGD-independent site onfibrinogen. Presented herein are data showing that the interactionbetween circulating leukocytes and endothelial cells is mediated by abridging effect of different parts of the fibrinogen molecule todistinct cell surface membrane receptors expressed on each cell type.

A. Demonstration of Fibrinogen Binding to HUVEC

1) Preparation of Iodinated Fibrinogen

Fibrinogen was iodinated using the Iodogen™ method. Briefly, Iodogen™was dissolved in dichloromethane for a final concentration of 1 μg/mland 170 μl of dissolved Iodogen™ that was dried in the bottom of a glasstube. Fibrinogen, prepared in Example 1A, was resuspended in 0.055 Msodium citrate buffer, pH 7.4, for a final concentration of 5 μg/ml. Twohundred μl of dissolved fibrinogen solution was placed into theIodogen™-coated tube with 700 μCi of carrier-free sodium iodide. Theadmixture was maintained on ice for 20 minutes with occasionalagitation. To stop the iodination reaction, the admixture was removedfrom the tube and gel filtered on a Sepharose™ G-25 coarse column(100×2.5). Fractions of iodinated fibrinogen were determined bytrichloroacetic acid precipitable counts. The labelled fibrinogenproduced was radiolabelled to a specific activity of 0.3 μCi/μg ofprotein. Labelled fibrinogen was used in the binding and inhibition ofbinding assays described herein at a concentration of 50 μg/ml whereasunlabelled fibrinogen was generally used at a concentration of 500μg/ml.

2) Analysis of Dose Dependency

To determine if fibrinogen bound to a cell surface HUVEC receptor and ifso, at what concentrations, increasing concentrations from 0.01micromolar (μM) up to 0.44 μM (0.14 μM is equivalent to 50 μg/ml; 0.29μM is equivalent to 100 μg/ml and 0.44 μM is equivalent to 150 μg/ml) ofiodinated fibrinogen (¹²⁵I-Fg) prepared above were separately admixed tomonolayers of HUVEC cells that were previously washed two times withserum-free RPMI 1640. The HUVEC cell cultures were initially plated inindividual wells of a 48 well plate coated for tissue culture (CostarCorp., Cambridge, Mass.) as described in Example 2A for culturing ofcells in T75 flasks. The divalent cation, presented as calcium chloride(CaCl₂), at a concentration of 2.5 mM was also admixed into thecell-fibrinogen admixtures. The inhibitor of fibrin polymerization,PPack (D-phenyl-1-prolyl-1 arginine chloramethyl; Calbiochem Boehring),was admixed to the cell admixtures at a concentration of 100 mM. PPackwas present in all assays where it was necessary to prevent thepolymerization of fibrinogen into fibrin.

The resultant admixtures were maintained at 22° C. for 45 minutes toallow for fibrinogen to bind to the plated HUVEC. After the maintenanceperiod, the cells were washed two times with serum-free RPMI 1640 toremove unbound fibrinogen. The cells were then solubilized in 10% SDSand the radioactivity associated under the maintenance conditions wasquantitated in a gamma counter.

The resultant data is plotted in FIG. 3 as ¹²⁵I-labelled fibrinogenbound in counts per minute (cpm) per well (X 10⁻³) on the Y-axis againstincreasing concentrations of ¹²⁵I-labelled fibrinogen (X 10⁻⁷ M) on theX-axis. The data shows that ¹²⁵I-labelled fibrinogen binds saturably ata concentration of approximately 0.36 μM to monolayers of unstimulatedHUVEC.

3) Analysis of Effect of HUVEC Stimulation by Exposure to TNF orLipopolysaccharide on Binding of Fibrinogen

To determine the effect that known stimulators of HUVEC have on thebinding characteristics of fibrinogen to HUVEC, dose-responseexperiments were performed as described in Example 3A2) on untreatedHUVEC and TNF or lipopolysaccharide (LPS, Genzyme))-stimulated HUVEC.TNF and LPS were separately admixed at the respective concentrations of5 nanograms (ng)/ml and 1.0 μg/ml to monolayers of HUVEC and maintainedat 37° C. for 4 hours prior to the admixture of the labelled fibrinogenranging in concentration from 0.01 μM up to 0.36 μM.

The resultant data is plotted in FIG. 4 as ¹²⁵I-labelled fibrinogenbound in molecules per cell (X 10⁻⁶) on the Y-axis against increasingconcentrations of ¹²⁵I-labelled fibrinogen (X 10⁻⁷ M) on the X-axis.Under stimulation with either TNF or LPS, the number of labelledfibrinogen molecules bound per cell doubled in comparison to those boundto unstimulated cells. Thus, the increase of fibrinogen binding toICAM-1 receptor on HUVEC is cytokine or immunostimulant mediated.

4) Analysis of Binding of D₃₀to HUVEC

Binding assays described above in Example 3A2) were also performed withthe fibrinogen homolog, D₃₀, to determine if that region of fibrinogenalso immunoreacted with HUVEC. Since D₃₀ was known to bind to leukocytesvia the Mac-1 receptor as described by Altieri et al., J. Biol. Chem.,265:12119-12122 (1990), these experiments were performed to determine ifregions of fibrinogen mediating the binding of leukocytes to HUVEC arecontained within the D₃₀ fragment. For this analysis, the D₃₀ fragmentof fibrinogen prepared in Example 1B was labelled with ¹²⁵I as describedfor labelling of fibrinogen above. Iodinated D₃₀ was admixed to HUVECmonolayers at a concentration of 10 μg/ml to form a binding complex.After washing the cells to remove the unbound D₃₀ as described inExample 3A2) above, the cells were solubilized and the amount of boundradioactivity was determined. The binding of D₃₀ to HUVEC was maximal at120 minutes of the maintenance period with approximately 60,000 cpm. Thebinding of D₃₀ was specifically competed by admixture of 50 fold molarexcess of cold fibrinogen thus confirming that D₃₀ specifically bound toa fibrinogen binding site on HUVEC. Myoglobin, a nonspecific protein,did not inhibit the binding of D₃₀ HUVEC.

Confirmation of the specificity of D₃₀ binding to HUVEC was obtained byinhibiting the binding of D₃₀ to ICAM-1 transfected cells prepared inExample 4 in the presence of the 14E11 IgG monoclonal antibody alsoprepared in Example 4. The inhibition of binding assays were performedas described in Example 5. The anti-ICAM-1 BD monoclonal antibodydescribed in Example 4 was also used in the assay. Both 14E11 and theanti-ICAM-1 BD monoclonal antibodies, at a concentration of 20 μg/ml inthe presence of MnCl₂, specifically inhibited the binding of D₃₀ toHUVEC. Approximately 3000 and 7500 cpm were recovered from the bindingof D₃₀ in the presence of CaCl₂ and MnCl₂, respectively, in the absenceof any inhibitors. With 14E11 and MnCl₂, D₃₀ binding to thetransfectants was completely inhibited. The portion of fibrinogencontaining D₃₀, therefore, binds to the fibrinogen receptor on HUVEC andto surface-expressed ICAM-1 on transfectants.

Furthermore, D₃₀-derived peptides defining the Mac-1 receptor bindingsite on D₃₀ did not block the binding of either fibrinogen or D₃₀ to theHUVEC, as described herein. Therefore, the bridging site of fibrinogenthat binds to the endothelial fibrinogen receptor is within the D₃₀fragment but is not the same region of D₃₀ that mediates the binding ofD₃₀ or fibrinogen to Mac-1 on leukocytes.

B. Demonstration of Fibrinogen Bridging the Binding of Mac-1-BearingCells to an RGD-Independent Fibrinogen Receptor on HUVEC

1) Analysis of Dose Dependency Over Time

In vivo, circulating leukocytes have been shown to bind to the apicalsurface of endothelial cells. In addition, experiments have beenperformed in vitro where the monocytic cultured cell line, THP-1 havingthe ATCC accession number TIB 202, (ATCC, Bethesda, Md.), was shown tobind directly to unperturbed HUVEC in the presence of divalent cationswith or without stimulation with 1 μM of the chemotactic peptide,N-formyl-methionyl-leucyl-phenylalanine (N-FMLP) (Sigma Chemical Co.) asdescribed by Altieri, J. Immunol., 147:1891-1898 (1991), herebyincorporated by reference. To determine whether this event was theresult of a fibrinogen mediated-bridging phenomenon, in vitro cellattachment binding assays were performed.

For these assays, HUVEC were plated in the medium described in Example2A at a density of approximately 1-5×10⁴ cells/well into flat-bottommicrotiter wells of a 96 well tissue-culture treated plate. The cellswere then washed in serum-free RPMI 1640 and further maintained with⁵¹-Chromium-labelled (⁵¹Cr) THP-1 cell suspensions previously exposed todifferent concentrations of unlabelled fibrinogen or left untreated. Tolabel THP-1 cells, serum-free suspensions of the cells at aconcentration of 1×10⁷ cells/ml were labelled with 0.5 mCi ⁵¹Cr (Na₂CrO₄having a specific activity of 487.4 mCi/mg, NEN Du Pont de Nemours) for2 hours at 37° C. with incorporation of an average of 12 to 20 cpm/THP-1cell. The labelled cells were then washed twice at room temperature withserum-free RPMI 1640 and resuspended in the same medium at aconcentration of 5 ×10⁵ cells/ml. The labelled cells were used in theassays within 2 hours from the labelling procedure. For the assays, thecells were pre-stimulated with 1 μM N-FMLP in the presence of 1 mM CaCl₂and 100 mM PPack. The resultant N-FMLP-stimulated THP-1 cell suspensionswere then separately admixed with the following: 1) Medium without anyadmixed fibrinogen as a control; 2) Fibrinogen at 1.2 mg/ml and at 2.5mg/ml concentrations; and 3) Normal human plasma (NHP) diluted 1:2 and1:50. Purified fibrinogen was prepared as described in Example 1.Fibrinogen was present in undiluted normal human plasma at theconcentration of approximately 1-3 mg/ml. The resultant admixtures weremaintained for 20 minutes at 22° C. to allow for the binding offibrinogen, either purified or present in NHP, to the Mac-1 receptor onthe surface of the THP-1 cells.

The resultant fibrinogen-bound THP-1 cells were then admixed to thewashed immobilized HUVEC described above to allow for the binding of anon-Mac-1 receptor binding site on fibrinogen to the RGD-independentfibrinogen receptor on the surface of HUVEC, thereby resulting in thebinding of THP-1 cells to HUVEC via a fibrinogen bridge. The admixtureswere maintained at 37° C. to allow for adhesion. At selected timeintervals between 1 to 60 minutes, the HUVEC monolayers were gentlywashed five times with serum-free RPMI 1640 to remove nonadherent orloosely adherent THP-1 cells to which fibrinogen was initiallyimmobilized. The adherent cells were then solubilized in 10% SDS and thecell lysate was quantitated in a beta scintillation counter. Spontaneous⁵¹Cr release from THP-1 cells was always less than 2% during theadhesion assay. The number of specifically attached THP-1 cells wasdetermined by dividing the cpm harvested by the cpm/cell.

The results of these experiments are shown in FIG. 5A and FIG. 5B wherethe data is expressed as numbers of ⁵¹Cr-labelled THP-1 cells (X 10⁻³)on the Y-axis plotted against the assay time on the X-axis. THP-1 cellsdid not significantly bind to immobilized HUVEC in the absence offibrinogen throughout the time course. In contrast, THP-1 cellsmaintained in the presence of 1.2 mg/ml fibrinogen exhibited significantincreases of binding to HUVEC over the time course with the maximum cellattachment occurring at 10,000 cells (FIG. 5A). In the presence of 2.5mg/ml of fibrinogen, TPC-1 cell attachment had not saturated at the endof 60 minutes where approximately 18,000 THP-1 cells were attached tofibrinogen (FIG. 5A). Similar binding curves were obtained in thepresence of NHP shown in FIG. 5B. Maximum THP-1 cell attachment ofapproximately 23,000 cells was obtained after 40 minutes in the presenceof NHP diluted 1:2 which contains approximately 0.5-1.5 mg/mlfibrinogen. NHP diluted 1:50 exhibited a profile comparable to that seenwith 1.2 mg/ml of purified fibrinogen. Thus, the time-dependent bindingof THP-1 cells to HUVEC was fibrinogen-dependent confirming thatfibrinogen, either purified or present in NHP, serves as a proteinbridge between the two cell types.

2) Analysis of Temperature Dependency

To determine the effects that temperature has on the ability offibrinogen to mediate the binding of ⁵¹Cr-labelled THP-1 cells tomonolayers of HUVEC, cell adhesion assays as described in Example 3B1)were performed at 22° C. and at 37° C. over the course of one hour. Theassays were performed in an identical manner as described above with theexception that 500 μg/ml of fibrinogen was used instead of 1.2 or 2.5μg/ml. The maximum binding of THP-1 cells to HUVEC at 22° C. was 7500cells after 40 minutes. At 60 minutes, the binding decreased to 5000cells. The data is significant in comparison to the approximately 2000THP-1 cells attached in the absence of fibrinogen. However, at 37° C.,approximately 20,000 THP-1 cells attached to HUVEC after 40 minutesagainst a background binding of approximately 5000 cells in the absenceof fibrinogen. The binding of ⁵¹Cr-labelled THP-1 cells to HUVEC, thus,is maximized at the physiologic temperature of 37° C. with physiologicconcentrations of fibrinogen.

3) Analysis of Cell Type Specificity

Cell adhesion binding assays described above were performed on bovineaortic endothelial cells (BAE) to determine if fibrinogen could mediatethe binding of ⁵¹Cr-labelled THP-1 cells to endothelial cells from adifferent source. Cell cultures were prepared from bovine aortafollowing procedures known to one skilled in the art. THP-1 cells wereeither left untreated or treated with 500 μg/ml fibrinogen over a 60minute time course. At selected time points, the cells were harvested asdescribed in Example 3B1) and the number of attached THP-1 cells weredetermined as previously described. In the absence of fibrinogen, THP-1cells did not significantly bind to BAE cells (less than 5000 cellsattached) after an initial rise of attachment peaking at 20 minutes.However, in the presence of fibrinogen, approximately 20,000 cells boundto BAE cells after a 40 minute maintenance period. This maximum bindingdropped off at 60 minutes to approximately 15,000 attached cells.Binding of THP-1 cells to HUVEC was done in parallel as a control forthe experiment; the non-saturable binding of THP-1 cells in the presenceof fibrinogen was maximal at 60 minutes with approximately 25,000 cellsattached. Thus, fibrinogen mediates the binding of THP-1 cells not onlyto human endothelial cells but also to bovine-derived endothelial cells.

4. Preparation of Anti-Endothelial Cell Monoclonal Antibodies to anRGD-Independent Fibrinogen Receptor on HUVEC

A. Preparation of Immunogen

HUVEC, cultured as described in Example 2A but without TNF or LPSstimulation, were prepared for use as immunogens in order to raisemonoclonal antibodies against HUVEC surface proteins for eventualscreening by assaying the inhibition of ¹²⁵I-labelled fibrinogen bindingto HUVEC cultures. For the immunizations, 10×10⁶ cells harvested fromthe culture plates by treatment with 4.0 mM EDTA and resuspended insaline were injected into mice as described below.

B. Preparation of Monoclonal Antibodies to an RGD-Independent FibrinogenReceptor on HUVEC

The HUVEC, prepared as immunogens according to Example 4A, were injectedintraperitoneally (i.p.) into separate BALB/c ByJ mice (The ScrippsResearch Institute Vivarium, La Jolla, Calif.). The mice receivedbooster injections at 1, 3 and 5 weeks. The last boost was 4 days priorto fusion.

The animals so treated were sacrificed and the spleen of each mouse washarvested. A spleen cell suspension was then prepared. Spleen cells werethen extracted from the spleen cell suspension by centrifugation forabout 10 minutes at 1000 rpm, at room temperature. Following removal ofthe resultant supernatant, the cell pellet was resuspended in 5 ml coldammonium chloride (NH₄Cl) lysing buffer, and was maintained for about 10minutes.

Ten ml of Dulbecco's Modified Eagle Medium (DMEM) (Whittaker M. A.Bioproducts) and Hepes buffer were admixed to the lysed cell suspensionto form an admixture, and that admixture was centrifuged for about 10minutes at 1000 rpm at room temperature.

After the resultant supernatant was decanted, the pellet was resuspendedin 15 ml of DMEM and Hepes and was centrifuged for about 10 minutes at1000 rpm at room temperature. The above procedure was repeated.

The pellet was then resuspended in 5 ml DMEM and Hepes. An aliquot ofthe spleen cell suspension was then removed for counting. Fusions wereaccomplished in the following manner using the non-secreting mousemyeloma cell line P3X63Ag8.653.1, a subclone of line P3X63Ag8.653 (ATCCAccession Number CRL 1580). With a myeloma to spleen cell ratio of about1 to 10 or about 1 to 5, a sufficient quantity of myeloma cells werecentrifuged into a pellet, washed twice in 15 ml DMEM and Hepes, andthen centrifuged for 10 minutes at 1000 rpm at room temperature.

Spleen cells and myeloma cells were combined in round bottom 15 mltubes. The cell mixture was centrifuged for 10 minutes at 1000 rpm. atroom temperature and the supernatant was removed by aspiration.Thereafter, 200 μl of 50 percent (weight per volume) aqueouspolyethylene glycol 4000 molecular weight (PEG) at about 37° C. wereadmixed with the pellet using a 1 ml pipette with vigorous stirring todisrupt the pellet. The cells were then gently mixed for between 15 and30 seconds. The resultant cell mixture was centrifuged 4 minutes at 700rpm.

At about 8 minutes from the time of adding the PEG, 5 ml of DMEM plusHepes buffer were admixed slowly to the pellet, without disturbing thecells. After 1 minute, the resulting admixture was broken up with a 1 mlpipette and was maintained for an additional 4 minutes. This admixturewas centrifuged for 7 minutes at 1000 rpm. The resultant supernatant wasdecanted, 5 ml of HT (hypoxanthine/thymidine) medium were slowly admixedto the pellet and the admixture was maintained undisturbed for 5minutes. The pellet was then broken into large chunks and the final cellsuspension was placed into T75 flasks (2.5 ml per flask) into which 7.5ml HT medium had been previously placed. The resulting cell suspensionwas maintained at 37° C. to grow the fused cells. After 24 hours, 10 mlof HT medium were admixed to the flasks followed 6 hours later byadmixture of 0.3 ml of 0.04 mM aminopterin. Forty-eight hours after thefusion, 10 ml of HAT (hypoxanthine/aminopterin/thymidine) medium wereadmixed to the flasks.

Three days after fusion, viable cells were plated out in 96-well tissueculture plates at about 2×10⁴ viable cells per well (768 total wells) inHAT buffer medium as described in Kennett et al., Curr. Top. Microbiol.Immunol., 81:77 (1978). The cells were fed seven days after fusion withHAT medium and at approximately 4-5 day intervals thereafter as neededwith HT medium. Growth was followed microscopically and culturesupernatants were collected about two weeks later.

C. Immunoscreening of Monoclonal Antibodies by Cell Adhesion Assays

The culture supernatants from HAT resistant cultures prepared above weresubsequently assayed for the presence of HUVEC RGD-independentfibrinogen receptor antibodies by the binding assays and cell adhesionassays respectively described in Example 3A and 3B, and furtherdescribed in Example 5. Culture supernatants were tested for theirability to inhibit the binding of ¹²⁵I-labelled fibrinogen to monolayersof HUVEC. For this assay, the monolayers were maintained in the presenceof hybridoma culture supernatant and 2.5 mM CaCl₂ for 30 minutes at 37°C. After the maintenance period, the supernatant-treated HUVEC werewashed once with RPMI 1640. Labelled fibrinogen was then admixed to thetreated HUVEC at a concentration of 50 μg/ml in the presence of 2.5 mMCaCl₂ and 100 mM PPack. The resultant admixtures were maintained for30-60 minutes at 22° C. The fibrinogen-treated HUVEC were then washed,solubilized and counted as described in Example 3A.

Supernatants were also screened for their ability to block the bindingof ⁵¹Cr-labelled THP-1 cells previously exposed to fibrinogen to HUVECcells. The assay was performed essentially as described for the celladhesion assay in Example 3B with the exception that the HUVEC wereseparately maintained with hybridoma supernatants for 30 minutes at 37°C. as described above. Following the antibody exposure, the cells werewashed once with culture medium prior to the admixture of thefibrinogen-bound and labelled THP-1 cells at the desired concentrationsas described in Example 3B.

Hybridoma culture supernatants that produced an antibody of thisinvention which effectively blocked the fibrinogen-mediated binding ofTHP-1 cells to monolayers of HUVEC were then selected for subsequentpurification and characterization. Hybridoma cultures producingantibodies against RGD-independent fibrinogen receptors (also referredto as a fibrinogen binding site) on HUVEC were identified. Four separateantibodies, designated 14E11, 16G8, 2E12 and 2B12, were obtained. 14E11was determined to be an IgG while the remaining monoclonals weredetermined to be IgMs. The monoclonal antibodies, specific for anendothelial cell RGD-independent fibrinogen receptor (also referred toas anti-ECR equivalent to anti-ICAM-1 based on the affinitychromatography analysis in Example 2), were shown to Immunoreact withHUVEC in addition to the purified ECR eluted from a fibrinogenSepharose™ column with EDTA as described in Example 2D, and to notimmunoreact with VNR.

D. Purification of the Selected Monoclonal Antibodies

The four hybridomas secreting anti-ECR antibodies as described inExample 4C were injected into 10-week old BALB/c mice as described belowto produce ascites fluid.

To that end, separate sets of 10-week old BALB/c mice were primed with0.3 ml of mineral oil and then injected intraperitoneally with 5×10⁶hybridoma cells for each monoclonal. The average time for development ofascites was 9 days. Following clarification by centrifugation at 15,000×g for 15 minutes at room temperature, ascites fluids produced byhybridomas were pooled and stored frozen at −20° C. to form monoclonalantibody compositions.

The ascites-produced monoclonal antibodies were further purified by fastprotein liquid chromatography (FPLC) using a Pharmacia Mono Q HR5/5anion exchange column (Pharmacia) using a 0-0.5 M NaCl gradient in 10 mMTris-HCl at pH 8.0 following directions supplied with the column. TheFPLC-treated monoclonal antibodies were then concentrated using anAmicon stirred ultrafiltration cell (Danvers, Mass.; PM 30 membrane) toa concentration of 1 mg/ml, dialyzed into PBS and stored at −70° C. toform purified MAb.

The monoclonal antibody, 14E11, was further affinity purified using aaffinity purification kit, Affi-Prep, according to the manufacturer'sinstructions (Bio-Rad, Richmond, Calif.). The IgM monoclonal antibodieswere further purified by hydroxylapatite gel filtration over a Bio-GelHPHT hydroxylapatite column according to manufacturer's instructions(Bio-Rad). These purified antibodies were used in subsequent bindingassays, Western immunoblots and immunoprecipitations are described inthe Examples.

E. Confirmation of the Immunospecificity of the Monoclonal Antibodies toan RGD-Independent Fibrinogen Receptor on HUVEC

The immunospecificity of the monoclonal antibodies, 14E11, 16G8, 2E12and 2B12, was confirmed by a number of approaches. Firstly, as describedin Example 2D, immunoprecipitation of the fibrinogen affinitychromatography-purified ECR was performed using 2E12 in comparison toimmunoprecipitation with a commercially available anti-ICAM-1 antibody(Becton Dickinson, referred to as BD). The 90-95 kD purified ECR wasimmunoprecipitated with both of the antibodies indicating that the 2E12antibody had the same immunospecificity as the anti-ICAM-1 antibody andthat the purified ECR was ICAM-1. The exact epitopes of the ECR (ICAM-1)recognized by the antibodies, 2E12 and Becton Dickinson anti-ICAM-1,have not been determined. Based on the blotting profile obtained byWestern immunoblots analysis described below and by the inhibition ofbinding data presented in Example 5, the epitopes are likely to beunique.

Fluorescent Activated Cellscan (FACscan) analysis was performed on anfibroblast-like cell line which was genetically engineered to expressthe recombinant form of ICAM-1 on the surface of the cells as describedby Seed et al., Nature, 331:624-627 (1988), hereby incorporated byreference. Briefly, a cDNA library, constructed using RNA prepared fromHL-60 cells induced with 12-0-tetradecanoyl phorbol 13-acetate (TPA),was transfected into COS cells. The cells expressing surface antigenswere screened by panning with anti-ICAM monoclonal antibodies,designated 8F5 and 84H10, resulting in the selection of a cDNA clone ina transfected cell expressing ICAM-1. These transfectants were used inFACscan analysis to confirm the immunospecificity of the anti-ECRantibodies of this invention.

For the analysis, 1×10⁶ ICAM-1 transfectant cells in suspension wereseparately admixed with 1 μg/ml affinity-purified 14E11, an admixture ofhydroxylapapatite purified of all three IgM monoclonal antibodies (16G8,2E12 and 2B12), with the anti-ICAM-1 BD monoclonal antibody and acontrol monoclonal antibody designated PMI-I having the ATCC AccessionNumber HB 9476. The latter recognizes the C-terminal hGPIIb fragment ofthe receptor GPIIb/IIIa found on platelets. The separate admixtures weremaintained for 30 minutes at 4° C. to form immunoreaction products.After 3 washes in serum-free RPMI 1640, the immunoreacted transfectedcells were admixed with fluorescein-conjugated goat anti-mouseimmunoglobulins and maintained for 30 minutes at 4° C. to form secondaryimmunoreaction products. After washing 3 times, the cells were subjectedto flow cytometry on a Becton Dickinson IV/40 fluorescence activatedcell sorter.

The results of the FACscan revealed that 16G8, 2E12 and 2B12 monoclonalantibodies specifically immunoreacted with the ICAM-1-expressingtransfectants comparably to that seen with the anti-ICAM-1 BD antibody.14E11, however; did not specifically immunoreact with theICAM-1-expressing transfectants as its profile overlapped that seen withthe control PMI-I antibody. Since the ICAM-1 expressed on thetransfectants is known to be unglycosylated, the lack ofimmunoreactivity of 14E11 with the cells is mostly likely due to thisreason. In the initial screen of the hybridomas, the 14E11 monoclonalantibody did block the binding of ¹²⁵I-labelled fibrinogen to HUVEC aswell as block the binding of THP-1 cells via a fibrinogen bridge toHUVEC cells.

That the 14E11 monoclonal antibody specifically recognized the ECRidentified as ICAM-1 was confirmed by Western immunoblot. Both HUVEC andDaudi cells were used for the blotting. Daudi is a Burkitt's lymphomahuman cell line that expresses high levels of ICAM-1 and is availablefrom ATCC having the ATCC Accession Number CCL 213. Cell lysates wereprepared from cultures of each cell type as described for preparation ofa cell lysate in Example 2A with the exception that the cells were lysedwith 0.5% Triton-X 100 and 0.5% NP-40. After centrifugation as describedin Example 2A, aliquots of each resultant supernatant wereelectrophoresed into multiple lanes by 10% SDS-PAGE.

Following the electrophoresis, the proteins in the gel were transferredelectrophoretically to nitrocellulose for subsequent immunoreactions.After the nitrocellulose blot was maintained for 2 hours at roomtemperature immersed in a solution of non-fat dry milk (Blotto) to blocknonspecific binding sites, it was then cut into strips to isolate eachindividual lane of electrophoresed proteins, 5 for Daudi and 8 forHUVEC. The nitrocellulose strips were then separately immunoreacted withtwo control antibodies, 2E1 and PMI-I, purified 14E11 IgG, 14E11 culturesupernatant and anti-ICAM-1 BD monoclonal antibody for 1 hour at roomtemperature to form primary immunoreaction products. The concentrationof the primary antibodies added was approximately 10 μg/ml. Theimmunoreacted blots were then washed 4 times with PBS for 5 minuteseach. The washed blots were then immersed for 1 hour at room temperaturein a solution of secondary ¹²⁵I-labelled goat anti-mouse antibodies(Zymed Laboratories Inc., San Francisco, Calif.) to form secondaryimmunoreaction products. The immunoreacted blots were then washed 4times with PBS and exposed to X-ray film for the detection ofimmunoreacted electrophoresed proteins from the cell lysatesupernatants. Additional controls included reacting the strips with aonly the labelled secondary antibody.

The results of the Western blot are shown in FIG. 6. The relativemolecular weights of the electrophoresed proteins in the cell lysatesupernatants were determined by comparison with a set of radiolabelledmolecular weight markers of 97, 66, 45, 30 and 21 kD shown in lane leftof the first set of 5 Daudi lanes and left of the second set of 8 HUVEClanes. Lanes designated 1-5 at the bottom of the blot for both Daudi andHUVEC were respectively immunoreacted with 2E1, PMI-I, affinity purified14E11, 14E11 culture supernatants and the anti-ICAM-1 BD monoclonalantibodies. A 90-95 kD band was detected in both Daudi and HUVEC celllysate supernatants with the affinity purified 14E11 monoclonal antibodyshown in number 3 labelled lanes. In addition, the affinity purified14E11 immunoreacted with a protein of approximately 50-55kD in lane 3 ofthe HUVEC-electrophoresed proteins. While the anti-ICAM-1 BD antibodyslightly immunoreacted with the Daudi proteins, it stronglyimmunoreacted with only the 90-95 kD band in the HUVEC electrophoresedproteins. The apparent difference in blotting patterns between the 14E11and anti-ICAM-1 BD monoclonal antibodies supports the position that theyrecognize separate epitopes on the ICAM-1 molecule. The control primaryantibodies, 2E1 and PMI-I, immunoreacted with the electrophoresed Daudiand HUVEC proteins as predicted based on prior characterizations ofbinding specificities. Thus, the 14E11 IgG monoclonal antibodyspecifically recognized the 90-95 kD cell surface protein isolated fromboth Daudi and HUVEC comparable to that seen with the anti-ICAM-1 BDantibody by both Western blot described herein and byimmunoprecipitation described in Example 2D.

In addition, as shown in Example 5, the affinity purified 14E11 antibodywas completely effective at inhibiting the binding of THP-1 cells viathe fibrinogen bridge to HUVEC.

5. Inhibition of Fibrinogen-Mediated Binding of Leukocytes toEndothelial Cells

A. Inhibition of Fibrinogen-Mediated Binding of THP-1 Cells to HUVECUsing Fibrinogen Analogs

1) Excess Cold Fibrinogen

Binding of ¹²⁵I-labelled fibrinogen binding to HUVEC assays wereperformed in the presence of 50 molar excess of unlabelled fibrinogen inorder to confirm the specificity of binding as shown in Example 3A. Thebinding assay was performed as described in Example 3A with theexception that unlabelled fibrinogen was admixed to HUVEC at a 50 molarexcess (approximately 7.5×10⁻³ M fibrinogen) concurrently with 50 μg/mlof ¹²⁵I-labelled fibrinogen. The protein-cell admixtures were maintainedat 22° C. for 30 minutes to allow for binding of fibrinogen to HUVEC. Inaddition to cold fibrinogen, a 50 fold molar excess of BSA wasseparately admixed as a control. The amount of fibrinogen bound underthe above conditions was quantitated as described in Example 3A. Theuninhibited total binding of ¹²⁵I-labelled fibrinogen saturated after 30minutes at approximately 4000 cpm per well. The BSA did not inhibit thebinding and thus ¹²⁵I-labelled fibrinogen exhibited a similar profile inthe presence of BSA. Unlabelled fibrinogen, however, competed thebinding of the labelled fibrinogen to half that of the total uninhibitedbinding. The binding of fibrinogen to the HUVEC surface fibrinogenreceptor is therefore specific.

The specificity of binding to an RGD-independent fibrinogen receptor wasconfirmed in competition assays performed as described above in thepresence of monoclonal antibodies against the beta subunit of theRGD-dependent VNR and in the presence of RGD-containing peptides. Forthe assays in which the ability of anti-VNR monoclonal antibodies,designated mAb 609 and mAb 7E3, to inhibit the binding of ¹²⁵I-labelledfibrinogen to HUVEC, the antibodies were separately maintained at aconcentration of 25 μg/ml with HUVEC for 20 minutes at 37° C. After themaintenance period, the antibody-treated HUVEC were washed once withserum-free RPMI 1640 to remove any unbound antibody. Labelled fibrinogenwas then admixed to the treated HUVEC at a concentration of 50 μg/ml inthe presence of 2.5 mM CaCl₂ and 100 mM PPack. The resultant admixtureswere maintained for 10-30 minutes at 22° C. The fibrinogen-treated HUVECwere then washed, solubilized and counted as described in Example 3A.

The results are shown in the bar graphs in FIG. 7 where the amount of¹²⁵I-labelled fibrinogen bound to HUVEC in cpm/well (X 10⁻³) is plottedon the Y-axis against the length of time labelled fibrinogen wasmaintained with HUVEC. Each part of the bar graph is separatelyidentified for each time point as follows:

total binding (no inhibitors admixed), +Fg (fibrinogen) admixed, +mAb609; and +mAb 7E3. Cold fibrinogen that was added in 50 molar excess asdescribed above almost completely inhibited the binding of ¹²⁵I-labelledfibrinogen to HUVEC. The VNR antibodies, however, had no inhibitoryeffect as anticipated by the results with the affinity chromatography inExample 2. Thus, fibrinogen binds to an receptor on HUVEC that is notVNR.

To confirm that the fibrinogen binding site on HUVEC was not RGDdependent, inhibition assays performed as described above for inhibitingwith antibodies except that they were done in the presence of 1.0 mM ofan RGD-containing peptide. A control RGE-containing peptide was alsoincluded in the assay. At selected time points over the time course ofone hour, the HUVEC were harvested as described in Example 3A.

The resultant data are shown in FIG. 8 where the amount of ¹²⁵I-labelledfibrinogen bound to HUVEC in cpm/well (X 10⁻³) is plotted on the Y-axisagainst the length of time labelled fibrinogen was maintained withHUVEC. Neither the RGD- nor the RGE-containing peptide had anyinhibitory effect of the binding of labelled fibrinogen to HUVEC ascompared to the total binding of the labelled fibrinogen in the absenceof any inhibitor. The RGD- and the RGE-containing peptide data are shownrespectively as the lines indicated by the solid and open squares. Coldfibrinogen completely inhibited the binding of labelled fibrinogen asexpected.

In summary, an excess of unlabelled fibrinogen completely inhibits thebinding of labelled fibrinogen to HUVEC and this binding is mediatedthrough an RGD-independent fibrinogen receptor that is not VNR.

2) Anti-ECR (ICAM-1) Monoclonal Antibodies

The monoclonal antibodies generated by immunizing mice with intactunstimulated HUVEC as prepared in Example 4 were tested for theirability to inhibit the binding of ¹²⁵I-fibrinogen to HUVEC eitherunstimulated or stimulated with TNF. The binding assays were performedas described in Example 3A with the exceptions described in Example 4for the screening of hybridoma culture supernatants. HUVEC were exposedto 5 ng/ml TNF for 4 hours at 37° C. prior to admixture of theantibodies. After the stimulation, the cells were washed once with theHUVEC culture medium prepared in Example 2A. The monoclonal antibodies,affinity purified 14E11, anti-ICAM-1 BD, and PMI-I (all described inExample 4), were separately admixed at a concentration of 20 μg/ml toeither stimulated or unstimulated HUVEC cultures and maintained for 30minutes at 37° C. After the antibody-reacted cells were washed once withculture medium, 50 μg/ml of ¹²⁵I-labelled fibrinogen prepared in Example3A was admixed to each well and maintained for 30 minutes at 22μC. Thecells were subsequently washed and solubilized as described in Example3A.

The resultant data is shown in FIG. 9A and FIG. 9B which respectivelyshown the effects of antibody exposure on binding of ¹²⁵I-labelledfibrinogen to unstimulated and TNF-stimulated HUVEC. The data isexpressed in a bar graph as the specific binding of ¹²⁵I-labelledfibrinogen in cpm/well (×10 −3) on the Y-axis against the specifictreatments on the X-axis. As shown in FIG. 9A, 14E11 partially inhibitedthe binding of fibrinogen while the BD anti-ICAM-1 antibody was slightlymore effective. However, in the TNF-stimulated HUVEC cultures shown inFIG. 9B, 14E11 completely inhibited the binding while the BD antibodywas only partially effective. The control antibody, PMI-I, was notinhibitory as compared to the amount of binding in the absence of anyantibody. The results, therefore, support the finding that 14E11specifically recognizes a fibrinogen receptor (or binding site) on thesurface of HUVEC the occupation of which completely inhibits the bindingof fibrinogen to TNF-stimulated HUVEC. This in vitro system mimics thatfound in vivo and thus the antibodies of this invention would be usefulas therapeutics.

6. Preparation of Anti-Fibrinogen Monoclonal Antibodies that Block theBinding of Fibrinogen to the RGD-Independent Fibrinogen Receptor onEndothelial Cells

A. Preparation of Immunogen

Fibrinogen and the fragments D and E prepared therefrom as described inExample 1, are prepared for use as immunogens in order to raisemonoclonal antibodies against the portion of fibrinogen that mediatesthe binding of fibrinogen to the RGD-independent fibrinogen binding siteon endothelial cells. For the immunizations, 50 μg of separatelyprepared fibrinogen immunogens are admixed in complete Freund's adjuvant(CFA),

B. Preparation of Monoclonal Antibodies to the Fibrinogen Site thatMediates the Binding of Fibrinogen on HUVEC

The HUVEC, prepared as immunogens according to Example 4A, are injectedintraperitoneally (i.p.) into separate BALB/c ByJ mice followed by asecond and third immunization using the same fibrinogen immunogens, eachabout three weeks apart, in incomplete Freund's adjuvant (IFA). The micereceive a boost of 50 μg of prepared fibrinogen immunogens intravenously(i.v.) in normal saline four days prior to fusion and a second similarperfusion boost one day later.

The animals so treated are sacrificed and the spleen of each mouse isharvested. A spleen cell suspension is then prepared and subjected tothe fusion protocol as described in Example 4. Growing clones are thenscreened for the expression of hybridomas having the selectedspecificity as described below.

C. Immunoscreening of Monoclonal Antibodies by Binding Assays

The culture supernatants from HAT resistant cultures prepared above aresubsequently assayed for the presence of fibrinogen antibodies thatfunction to inhibit the binding of fibrinogen to the RGD-independentfibrinogen receptor on endothelial cells. The supernatants are screenedby both the inhibition of ¹²⁵I-labelled fibrinogen binding assay and theinhibition of ⁵¹cr-labelled and fibrinogen-bound THP-1 cell attachmentassay as described in Example 4C, which are based on the assaysdescribed in Example 3A and 3B. The inhibition of binding assay isfurther described in Example 5.

Hybridoma culture supernatants that produce an antibody that binds tothe site on fibrinogen that mediates the binding of fibrinogen to theRGD-independent fibrinogen receptor on endothelial cells (also referredto as ICAM-1 based on the analysis in Examples 2-5 are then selected forsubsequent purification and characterization. Hybridoma culturesproducing antibodies against fibrinogen are identified. The monoclonalantibodies, specific for a region on fibrinogen that binds to theRGD-independent fibrinogen receptor on endothelial cells (ICAM-1) shownto immunoreact with fibrinogen in addition to the analogs thereof, andto not immunoreact with the site on fibrinogen that mediates the bindingto MAC-1, namely the site defined by the D₃₀-derived peptides describedin Example 3.

D. Purification of the Selected Monoclonal Antibodies

The selected hybridomas secreting anti-fibrinogen antibodies asdescribed in Example 6C are injected into 10-week old BALB/c mice asdescribed in Example 4D produce ascites fluid.

The ascites-produced monoclonal antibodies are further purified by fastprotein liquid chromatography (FPLC) using a Pharmacia Mono Q HR5/5anion exchange column (Pharmacia) using a 0-0.5 M NaCl gradient in 10 mMTris-HCl at pH 8.0 following directions supplied with the column. TheFPLC-treated monoclonal antibodies are then concentrated using an Amiconstirred ultrafiltration cell (Danvers, Mass.; PM 30 membrane) to aconcentration of 1 mg/ml, dialyzed into PBS and stored at −70° C. toform purified monoclonal antibody.

The monoclonal antibodies is further affinity purified using a affinitypurification kit, Affi-Prep, according to the manufacturer'sinstructions (Bio-Rad, Richmond, Calif.). The IgM monoclonal antibodiesare further purified by hydroxylapatite gel filtration over a Bio-GelHPHT hydroxylapatite column according to manufacturer's instructions(Bio-Rad). These purified antibodies are used in subsequent bindingassays, Western immunoblots and immunoprecipitations for use in thisinvention.

7. Preparation of Anti-Endothelial Cell Monoclonal AntibodiesImmunoreactive With the RGD-Independent Fibrinogen Receptor on DaudiCells

A. Preparation of Immunogen

Daudi is a Burkitt's lymphoma human cell line that expresses high levelsof ICAM-1 and is available from ATCC having the ATCC Accession NumberCCL 213. Daudi was prepared for use as immunogens in order to raisemonoclonal antibodies against Daudi surface proteins for eventualscreening by assaying the inhibition of fibrinogen-mediated binding ofleukocytes to HUVEC.

Daudi was cultivated in complete RPMI 1640 medium supplemented with 10%fetal calf serum (FCS, Whittaker) and 2 mM L-glutamine (IrvineScientific, Santa Ana, Calif.).

B. Preparation of Monoclonal Antibodies to a Fibrinogen Receptor onDaudi

For the immunizations, 10×10⁶ Daudi cells harvested from the cultureplates by treatment with 4.0 mM EDTA and resuspended in saline wereinjected into mice and monoclonal antibodies prepared as described inExample 4B.

C. Immunoscreening of Monoclonal Antibodies by Binding Assays

The culture supernatants from HAT resistant cultures prepared above wereassayed for HUVEC fibrinogen receptor antibodies by flow cytometry asdescribed by Languino, et al., Cell, 73:1423-1434 (1993). HUVEC wereactivated with TNF to increase the number of fibrinogen binding sitesexpressed on the surface of the cells as described in Example 3A3.Briefly, TNF was admixed at 5 nanograms (ng)/ml and added to monolayersof HUVEC and maintained at 37° C. for 4 hours. Aliquots of suspended TNFstimulated HUVEC at 1.5×10⁷/ml were washed in PBS, pH 7.2, plus 5 mMEDTA; blocked with 20% normal human serum to prevent Fc-mediatedmonoclonal antibody binding for 30 min at 4° C.; and incubated withaliquots of the culture supernatants containing the monoclonal antibodyfor 30 min at 4° C. in complete RPMI 1640 medium plus 10% FCS. Afterwashes, binding of the primary monoclonal antibodies was revealed by theaddition of a 1:20 dilution of fluorescein-conjugated goat anti-mouseF(ab′)₂ fragments for 30 min at 4° C. Cells were washed and immediatelyanalyzed on a Becton-Dickinson IV/40 fluorescence activated cell sorter.Background fluorescence was assessed in the presence of controlanti-plasminogen monoclonal antibody.

Hybridoma culture supernatants that produced an antibody which bound tosurface proteins on TNF stimulated HUVEC were identified. Three separateantibodies, designated 1G12, 2D5, and 6E6 were obtained. Theseantibodies were subsequently assayed for their ability to blockfibrinogen-mediated binding of leukocytes (THP-1) to HUVEC as describedin Example 3B. Hybridoma culture supernatants that produced an antibodywhich effectively blocked the fibrinogen-mediated binding of THP-1 cellsto monolayers of HUVEC were selected for subsequent purification asdescribed in Example 4D and further characterized. Two separateantibodies, designated 1G12 and 2D5, were obtained. A third antibody,6E6, which reacted with the TNF stimulated HUVEC but did not blockfibrinogen-mediated binding of leukocytes to HUVEC was also purified asdescribed in Example 4D and characterized.

D. Identification of Fibrinogen Binding Site on HUVEC as ICAM-1

Purified monoclonal antibodies 1G12, 2D5, and 6E6 were furthercharacterized to identify the fibrinogen binding site on HUVEC byreactivity with wild-type CHO cells and transfected CHO cells whichexpressed recombinant ICAM-1 on their surface.

1) Preparation of Cells

The Chinese Hamster Ovary (CHO) cells were cultivated in DMEM medium(Whittaker) supplemented with 10% fetal calf serum (FCS, Whittaker) and1 mM L-glutamine (Irvine Scientific, Santa Ana, Calif.), non-essentialamino acids, penicillin-streptomycin.

2) Transfection of ICAM-1 Expression Vector Into CHO Cells

A full length cDNA clone encoding for ICAM-1 (Simmons et al., Science,331:624-627 (1988)) was inserted into the mammalian expression vectorpRC/CMV (Invitrogen, San Diego, Calif.), oriented, and transfected (15μg plasmid DNA) in subconfluent cultures of CHO cells byelectroporation. Forty-eight hours after transfection, CHO cells wereharvested and diluted to 6×10³ cells/80 mm diameter tissue culture Petridish (Costar) in DMEM (Whittaker) selection medium containing 10% FCS, 1mM L-glutamine, non-essential amino acids, penicillin-streptomycin plus1 mg/ml G418 (Geneticin, GIBCO, Grand Island, N.Y.). After a 2 to 3 weekculture in selection media, CHO cells were harvested and phenotypicallycharacterized by flow cytometry using anti-ICAM-1 monoclonal antibody(Becton Dickinson, referred to as BD or LB-2). CHO cells which werereactive with the anti-ICAM-1 were cloned by limiting dilution in96-well tissue culture plates (Costar) in DMEM selection medium at 0.5-2cells/well.

3) Immunoreactivity of Monoclonal Antibodies with CHO Cells

The monoclonal antibodies 1G12, 2D5, and 6E6 were assayed for reactivitywith wild-type and ICAM-1 transfected CHO cells. Briefly, aliquots ofsuspended CHO cells at 1.5×10⁷/ml were washed in PBS, pH 7.2, plus 5 mMEDTA: blocked with 20% normal human serum to prevent Fc-mediatedmonoclonal antibody binding for 30 min at 4° C.; and incubated withaliquots of each monoclonal antibody for 30 min at 4° C. in completeRPMI 1640 medium plus 10% FCS. After washes, binding of the primarymonoclonal antibodies was revealed by the addition of a 1:20 dilution offluorescein-conjugated goat anti-mouse F(ab′)₂ fragments for 30 min at4° C. Cells were washed and immediately analyzed on a Becton-DickinsonIV/40 fluorescence activated cell sorter. Background fluorescence wasassessed in the presence of control anti-plasminogen monoclonalantibody.

Results of the FACScan revealed that the monoclonal antibodies 1G12,2D5, and 6E6 specifically immunoreacted with the ICAM-1-expressingtransfectants but not the wild-type CHO cells indicating that themonoclonal antibodies reacted specifically with the recombinant ICAM-1.

4) Immunoscreening of Monoclonal Antibodies by Binding Assays

The monoclonal antibodies 1G12, 2D5, and 6E6 which recognize ICAM-1 weresubsequently assayed for their ability to inhibit fibrinogen-mediatedleukocyte binding to HUVEC. The monoclonal antibodies are screened bythe inhibition of ⁵¹Cr-labelled and fibrinogen-bound THP-1 in a cellattachment assay as described in Example 4C, which are based on theassays described in Example 3A and 3B. The inhibition of binding assayis further described in Example 5.

The monoclonal antibodies 1G12 and 2D5, specific for a region on ICAM-1on endothelial cells (ICAM-1), inhibited the fibrinogen-mediatedleukocyte binding to HUVEC. The monoclonal antibody 6E6, specific for aregion on ICAM-1 on endothelial cells (ICAM-1), did not inhibit thefibrinogen-mediated leukocyte binding to HUVEC.

8. Characterization of the I-CAM Receptor Binding Site on Fibrinogen

A. Polypeptide Synthesis

Polypeptides derived from the γ chain of fibrinogen were synthesizedusing the classical solid-phase technique described by Merrifield, Adv.Enzymol., 32:221-296 (1969) as adapted for use with a model 430automated peptide synthesizer (Applied Biosystems, Foster City, Calif.).The amino acid residue sequence of the synthesized polypeptide γ3 isN¹¹⁷NQKIVNLKEKVAQLEA¹³³ (SEQ ID NO 2). The residue numbers correspondingto specific regions in the mature γ chain are shown for the γ3polypeptide. The amino acid residue sequence of the synthesizedpolypeptide L10 is L⁴⁰²GGAKQAGDV⁴¹¹ (SEQ ID NO 5). The residue numberscorresponding to specific regions in the mature γ chain are shown forthe L10 polypeptide. Prepared polypeptide resins were cleaved byhydrogen fluoride, extracted and analyzed for purity by high-performanceliquid chromatography (HPLC) using a reverse-phase C18 columnmanufactured by Waters Associates, Milford, Mass. The amino acidcomposition was verified as described by Altieri, et al., J. Biol.Chem., 268:1847-1853 (1993).

1) Binding Assay with Iodinated γ3 and Cells

Ninety-six well assay plates (Costar, Cambridge, Mass.) were coated withincreasing concentrations (1-100 μg/ml) of the polypeptides γ3 and L10in PBS, pH 7.2, for 18 h at 4° C. Wells were washed in PBS, pH 7.2 andblocked with 3% gelatin for 60 min at 37° C.

Serum-free suspensions of JY lymphocytes were labeled with 0.5 mCi ⁵¹Cr(Na₂CrO₄, specific activity 487.4 mCi/mg, E. I., DuPont de Nemours,Wilmington, Del.) for 2 h at 37° C., with a final incorporation of 0.2-4cpm/cell (Languino, et al., supra). Aliquots of ⁵¹Cr-labeled JYlymphocytes at 1×10⁶/ml were added to peptide-coated wells for 45 min at37° C., washed, solubilized in 20% SDS, and radioactivity associatedunder the various conditions was determined in a scintillationbeta-counter. The number of attached cells was quantitated by dividingthe cpm harvested by the cpm/cell. Data are the mean ± S.D. of twoindependent experiments.

The specific binding of radiolabelled ICAM-1⁺ JY lymphocytes toincreasing concentrations of γ3 was measured to characterize the ICAM-1binding site. The results of the binding assay are shown in FIG. 12. Thenumber of attached cells is plotted on the Y-axis against peptideconcentration (μg/ml) plotted on the X-axis. The results demonstratethat JY lymphocytes strongly adhered to immobilized γ3 in a specific anddose-dependent fashion, while control peptide L10 did not supportlymphocyte adhesion at any concentration tested under the sameexperimental conditions.

B. Competition of Binding of γ3 Polypeptide to ICAM-1 Binding Site on JYLymphocytes

1) Inhibition of γ3 Binding to ICAM-1⁺ JY Lymphocytes With Anti-ICAM-1Monoclonal Antibodies

The specificity of the γ3 polypeptide for the ICAM-1 receptor on JYlymphocytes was confirmed in assays where the binding of purified γ3 toJY lymphocytes was inhibited by monoclonal antibodies which recognizeICAM-1 and have been shown to block ICAM-1:fibrinogen interaction butnot by monoclonal antibodies which recognize ICAM-1 but have not beenshown to block ICAM-1:fibrinogen interaction.

Binding inhibition assays were performed using radiolabeled ICAM-1⁺ JYlymphocytes and the γ3 polypeptide. The competitive inhibitors evaluatedin the assay included the following: monoclonal antibodies 1G12 and 2D5which recognize ICAM-1 and have been shown to block ICAM-1:fibrinogeninteraction (Languino et al., Cell, 73:1423-1434 (1993)), monoclonalantibody 6E6 which recognizes ICAM-1 and has not been shown to blockICAM-1:fibrinogen interaction, and monoclonal antibody 6A11 whichrecognizes EPR-1. The peptides used in this assay were the γ3 andirrelevant polypeptide L10 prepared as described in Example 7A.

Ninety-six well assay plates were coated with 20 μ/ml of thepolypeptides γ3 and L10 and the ⁵¹Cr-labeled JY lymphocytes wereprepared as described above. The ⁵¹Cr-labeled JY lymphocytes werepreincubated with saturating concentrations (25 μg/ml) of monoclonalantibodies anti-ICAM-1 1G12, 2D5, or 6E6; or anti-EPR-1 6A11 beforeincubation in the 3 peptide-coated plates. Binding of radiolabelled JYlymphocytes was measured after a 45 min maintenance period in thepresence of the monoclonal antibodies in the γ3 peptide-coated plate.Background binding was assessed by the incubation of ⁵¹Cr-labeled JYlymphocytes which had not been preincubated with a monoclonal antibodyin the L10 peptide-coated plate. Data are the mean ± S.D. of replicatesof two independent experiments.

The results of the inhibition of binding assays are shown in FIG. 12.The number of attached cells is plotted on the Y-axis against thepeptide L10 plotted on the X-axis. Also plotted on the X-axis is thepeptide γ3 and the monoclonal antibodies used in the inhibition study.The amount of inhibition by the individual competitors is consistentwith the postulated recognition of fibrinogen (Languino, et al., supra)by anti-ICAM-1 monoclonal antibodies 1G12 and 2D5 which werespecifically selected for their ability to block ICAM-1:fibrinogeninteraction. 1G12 and 2D5 completely inhibited the adhesion of JYlymphocytes to γ3-coated plates. The control anti-ICAM-1 monoclonalantibody 6E6 which recognizes ICAM-1 and has not been shown to blockICAM-1:fibrinogen interaction, did not inhibit adhesion of JYlymphocytes to γ3-coated plates. The irrelevant polypeptide, L10, didnot support significant JY lymphocyte adhesion under the sameexperimental conditions. Thus, the minimal amino acid residue sequencerequired to promote the attachment of the γ chain of fibrinogen toICAM-1 is from amino acid residue 117 to amino acid residue 133 asdetermined from the complete inhibition of binding of ICAM-1⁺ JYlymphocyte cells to γ3 by monoclonal antibodies which recognize ICAM-1and have been shown to block ICAM-1:fibrinogen interaction.

C. Binding Assay of γ3 Polypeptide to Transfected Chinese Hamster OvaryCells Expressing ICAM-1

The direct physical interaction of γ3 with ICAM-1 expressed on thesurface of CHO cells was investigated.

1) Preparation of Cells

The Chinese Hamster Ovary (CHO) cells were cultivated and as describedin Example 8Cl. Quantitation of ICAM-1 surface expression on wild-type(WT) CHO cells or on ICAM-1 transfectants was carried out withmonoclonal antibodies LB-2 (Clark et al., Human Immunol., 16:100-113(1986)) or 2D5 (identified in Example 7C). Background fluorescence wasdetermined in the presence of control monoclonal antibody OKM1.

2) Preparation of Labeled Polypeptides

A variant polypeptide γ3 (KYG-γ3) duplicating the fibrinogen γ chainsequence 117-133 N¹¹⁷NQKIVNLKEKVAQLEA¹³³ (SEQ ID NO 2) was synthesizedwith the addition of Lys-Tyr-Gly residues at the amino terminus tofacilitate radiolabeling of the polypeptide (KYGN¹¹⁷NQKIVNLKEKVAQLEA¹³³,SEQ ID NO 4). The variant γ3 and L10 polypeptides were synthesized asdescribed in Example 7A. Typically, 2 mg of KYG-γ3 were iodinated with 5mCi ¹²⁵I-Na (Amersham, Arlington Heights, Ill.) by the IODO-GEN method(Fraker et al., Biochem Biophys Res Commun., 80:849-857, (1978)) for 45min at 4° C. Free radioactivity was separated from polypeptide-boundradioactivity by gel-filtration over a Bio-Gel P-2 column (Bio-Rad,Richmond, Va.) which had been preequilibrated with PBS, pH 7.2,containing 0.01% BSA, with a flow rate of 0.2 ml/sec.

3) Binding Assays of Polypeptides to CHO Cells Expressing ICAM-1

Confluent monolayers of ICAM-1 CHO transfectants or wild type (WT) CHOcells were incubated in serum-free RPMI 1640 medium with increasingconcentrations of ¹²⁵I-labeled γ3 peptide (10-150 μg/ml) in the presenceof 1 mM CaCl₂ and 1 mM MgCl₂ for 45 min at 22° C. Cells were washed withthree rapid changes of serum-free RPMI 1640, solubilized in 10% SDS, andradioactivity associated under the various conditions tested wasquantitated in a beta-counter. Non specific binding was assessed in thepresence of a 100-fold molar excess unlabeled γ3 peptide, or controlpeptide L10 added at the start of incubation, and was subtracted fromthe total as described above.

The specific binding of γ3 to CHO cells expressing ICAM-1 and WT CHOcells was measured to characterize the direct physical interaction of γ3with ICAM-1. The results of the binding assay are shown in FIG. 13A. Theng of ¹²⁵I-γ3 bound per well is plotted on the Y-axis against thepeptide concentration (μg/ml) plotted on the X-axis. The resultsdemonstrate that ¹²⁵I-labeled γ3 polypeptide bound to ICAM-1 CHOtransfectants in a specific and dose-dependent fashion. ¹²⁵I-labeled γ3polypeptide did not bind to WT CHO cells at 50 μg/ml, under the sameexperimental conditions (FIG. 13B). Binding of the ¹²⁵I-labeled γ3polypeptide was competitively inhibited by a 100-fold molar excessunlabeled γ3, but not by comparable concentrations of controlpolypeptide L10 (not shown).

9. Preparation of a Monoclonal Anti-γ3 Polypeptide Specific Antibody

A. Preparation of Immunogen

For preparation of a polypeptide immunogen, the synthetic polypeptide γ3(N¹¹⁷NQKIVNLKEKVAQLEA¹³³, SEQ ID NO 2) is prepared as described inExample 7A but is modified with a carboxy-terminal cysteine(N¹¹⁷NQKIVNLKEKVAQLEA¹³³C, SEQ ID NO 3). The synthesized γ3 is coupledto keyhole-limpet-hemocyanin (KLH) (Sigma, St. Louis, Mo.) using theheterobifunctional crosslinking agent,N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (PierceBiochemicals, Rockford, Ill.). For the coupling procedure, 80microliters (μl) of 10 mg/ml SPDP dissolved in dimethylformamide isadmixed dropwise to 400 μl 15 mg/ml KLH in 0.1 M phosphate, 0.1 M NaClat pH 8.5 under continuous stirring conditions for 30 minutes at 22° C.in order to form SPDP-activated KLH. The resultant SPDP-activated KLH isthen extensively dialyzed at 4° C. against a buffered solution of 0.1 Mphosphate and 0.1 M NaCl at pH 7.4 in order to remove uncoupled SPDP.Six mg of prepared γ3 having a C-terminal cysteine is first dissolved in2 ml of 0.1 M phosphate and 0.1 M NaCl at pH 7.4 and then admixed withSPDP-activated KLH prepared above under continuous stirring conditions.The degree of coupling of γ3 with KLH is monitored by thepyridine-2-thione release at 343 nm (ε: 8.08×10³ M⁻¹ cm⁻¹) in aspectrophotometer.

B. Preparation of Anti-γ3 Monoclonal Antibodies

Balb/c ByJ mice (Scripps Clinic and Research Foundation Vivarium, LaJolla, Calif.) are immunized and monoclonal antibodies prepared asdescribed in Example 4B. Culture supernatants are collected about twoweeks later and assayed for the presence of γ3— specific antibody bysolid phase radioimmunoassay (RIA).

Briefly, 50 μl of PBS containing 5 μg/ml of the prepared γ3 immunogen isadmixed into the wells of microtiter plates. The plates are maintainedovernight (about 16 hours) at 4° C. to permit the γ3 immunogen to adhereto well walls. After washing the wells four times with SPRIA buffer(2.68 mM KCl, 1.47 mM KH₂PO₄, 137 mM NaCl, 8.03 mM Na₂HPO₄, 0.05%Tween-20, 0.1 KIU/ml Traysol, 0.1% bovine serum albumin (BSA), 0.015%NaN₃), 200 μl of SPRIA buffer containing 3% normal goat serum (NGS) and3% BSA are admixed to each well to block excess protein binding sites.The plates are maintained for 30 minutes at 20° C., the wells emptied byshaking, and blotted dry to form a solid-support, i.e., a solid matrixto which γ3 immunogen is operatively affixed.

To each well is then admixed 50 μl of hybridoma tissue culturesupernatant to form a solid-liquid phase immunoreaction admixture. Theadmixture is maintained for 2 hours at 37° C. to permit formation ofsolid-phase immunoreaction products. After washing the wells asdescribed above, 50 μl of ¹²⁵I-labelled goat anti-mouse IgG at 0.25 μgprotein/ml are admixed to each well to form a labelling reactionadmixture. Radioiodination of immunochemically purified goat anti-mouseIgG is performed enzymatically utilizing the IODO-GEN iodinationprocedure as described in Example 3A1. The resultant admixture ismaintained for one hour at 37° C. to permit formation of ¹²⁵I-labelledsolid-phase immunoreaction products. After washing the wells asdescribed above, the amount of ¹²⁵I-labelled product bound to each wellis determined by gamma detection.

Hybridomas are selected from hybridoma cultures that secrete anti-γ3antibodies into their culture media, and further characterized asdescribed herein.

C. Production and Purification of anti-γ3 Monoclonal Antibodies

Hybridoma anti-γ3 is cultured in a 5% CO₂, humidified atmosphere at 37°C. in DMEM containing 2 mM L-glutamine, 50 μg/ml gentamicin, 10% fetalbovine serum, 10% horse serum, all from Grand Island Biological Co.,Lawrence, Mass., 10% NCTC medium from Microbiological Associates,Rockville, Md., 1 mM hypoxanthine and 0.3 mM thymidine, both from SigmaChemical Corp., St. Louis, Mo. Cell concentration is kept in the rangeof about 1-2×10⁵ cells per ml of medium to about 1-2×10⁶ cells per ml ofmedium for cell growth, division, and production of antibody.

To produce ascites tumor fluid containing anti-γ3 antibody molecules,10-week old Balb/c mice are immunologically primed by intraperitonealinjection with 0.3 ml of mineral oil and subsequently intraperitoneallyinjected with 3-5×10⁵ anti-γ3 hybridoma cells. The inoculated mice arethen maintained for a time period sufficient for anti-γ3antibody-containing ascites tumor fluids to accumulate, e.g., for about10 to about 21 days. The ascites fluid is collected and clarified bycentrifugation at 15,000×g for one hour at 4° C. and stored frozen at−20° C.

Anti-γ3 antibody molecules are isolated from the ascites fluid bysubjecting the fluid to fast protein liquid chromatography (FPLC) on aPharmacia Mono QHR 5/5 anion exchange column in a Pharmacia FPLC System(both from Pharmacia) using a 0-0.5 M NaCl gradient in 10 mM Tris-HCl,pH 8.0, and following the directions supplied with the column. Theanti-γ3 antibody molecules so isolated can then be transferred to anyphysiologically tolerable diluent desired by dialysis.

Alternatively, anti-γ3 antibody molecules can be isolated from theascites tumor fluid by precipitation with ammonium sulfate according tothe method described by Goding, Monoclonal Antibodies: Principles andPractice, Academic Press, p100-101 (1983). Briefly, that method entailsslowly admixing saturated ammonium sulfate to the ascites fluid untilabout a 45% to about a 50% ammonium sulfate concentration is achieved.The precipitated immunoglobulins are then collected by centrifugation at2000×g, preferably 10,000×g. The precipitate is washed 2 or 3 times in40% saturated ammonium sulfate. The precipitated anti-γ3 antibodymolecules are then dialyzed against 500-1000 volumes of phosphatebuffered saline (PBS) or any other physiologically tolerable diluentdesired to remove ammonium sulfate. The dialysis fluid is changedseveral times at intervals of a few hours. The protein concentration ofthe recovered dialyzed anti-γ3 antibody solution is determined by theLowry method [Lowry et al., J. Biol. Chem., 193, 265-275 (1951)] using abovine serum albumin standard.

The anti-γ3 antibodies produced are capable of immunoreacting with theimmunizing γ3 polypeptide and with fibrinogen, but do not producedetectable immunoreaction above, background when using fibrinogenpolypeptide derived from other regions of fibrinogen, such as the Mac-1binding site, e.g., a polypeptide defined by residues 190-202 shown inSEQ ID NO 1.

10. Effect of Plasma Adhesive Proteins on Leukocyte-EndotheliumInteraction

The potential role of the plasma adhesive proteins fibrinogen,vitronectin, and fibronectin on mediating leukocyte-endotheliuminteraction was investigated. Fibrinogen had previously been shown toincrease leukocyte-endothelium interaction (Languino et al., supra).Since two additional plasma proteins, vitronectin and fibronectin, sharefibrinogen's adhesive properties, their role in the regulation ofcell-cell interaction was investigated. The functional adhesiveproperties of the plasma proteins were determined in cell attachmentassays.

A. Purification of Fibrinogen, Vitronectin, and Fibronectin

Fibrinogen was isolated from fresh plasma and purified fromcontaminating fibronectin as described in Example 1A. Human fibronectinwas purified from plasma as described in Engvall and Ruoslahti, Int. J.Cancer, 10:1-5 (1977). Human vitronectin was purchased from GIBCO(Bethesda, Md.) or donated by Helena Hessle (Telios Pharmaceuticals, SanDiego, Calif.). Control proteins transferrin or BSA were purchased fromSigma (St. Louis, Mo.).

B. Preparation of Cells

1) Cell Cultivation

The promyelocytic HL-60 (Collins, Blood, 70:1233-1244 (1987) werecultivated in the same manner as HUVEC in Example 2A. Suspensions ofHL-60 were terminally differentiated to a monocyte-like phenotype by a 4to 5 day culture in the presence of 0.1 μM 1,25 dihydroxy vitamin D₃(BioMol, Plymouth Meeting, Pa.) and 17.8 μg/ml indomethacin (Calbiochem,San Diego, Calif.) as described by Collins, supra. The monocytic cellline THP-1 (Tsuchiya et al., Int. J. Cancer, 26:171-176 (1980)) wascultivated in the same manner as HUVEC in Example 2A with the additionof 10⁻⁵ M β-mercaptoethanol (Eastman Kodak, Rochester, N.Y.).

2) Phenotypic Characterization of Terminally-Differentiated HL-60 Cells

The phenotypical changes in cell surface adhesion receptors duringmonocytic differentiation of HL-60 cells were quantitated by flowcytometry as described in Languino, et al supra and Example 7C. Themonoclonal antibodies used in the assay included the following: BK (alsoknown as LB-2; Becton Dickinson) which recognizes ICAM-1, IB4 whichrecognizes the β₂ integrin subunit CD18 (Wright et al., Proc. Natl.Acad. Sci. U.S.A., 85:7734-7738 (1988)), LM609 which recognizes α_(v)β₃,OKM1 which recognizes CD¹¹b (Cheresh, Proc. Natl. Acad. Sci. U.S.A.,84:6471-6475 (1987), and 142 which recognizes α_(v) (D. A. Cheresh, TheScripps Research Institute, San Diego, Calif.).

Results indicated minimal changes in the surface density of the β₂integrin subunit CD18 detected by IB4 and α_(v) detected by 142, anapproximate 15-20 fold increase of the α subunit (α_(M), CD11b) of theleukocyte fibrinogen receptor CD11b/CD18 detected by OKM1, a 6-20 foldincrease in α_(v)β₃ integrin as detected by LM609 but with considerableheterogeneity, and an approximate 10 fold increase in ICAM-1 as detectedby BD.

C. Effect of Fibrinogen Vitronectin, and Fibronectin onLeukocyte-Endothelial Interaction

1) Binding Assays

The effect of fibrinogen, vitronectin, and fibronectin on theinteraction of terminally-differentiated HL-60 and monocytic THP-1 cellswith HUVEC was determined as described in Languino, supra. Briefly,serum-free suspensions of HL-60 or THP-1 cells were labeled with ⁵¹Cr asdescribed in Example 81A to a final incorporation of 0.2-4 cpm/cell.After washes in PBS, pH 7.2, aliquots of the HL-60 cells wereresuspended in serum-free RPMI 1640 at 1.5×10⁶/ml and stimulated with 10μM of the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP,Sigma, St. Louis, Mo.) and equilibrated with 150 μ/ml fibrinogen,vitronectin, or fibronectin in the presence of 1 mM CaCl₂ for 20 min at22° C. Equilibrated HL-60 were added to resting HUVEC monolayers thathad been previously incubated with fresh complete culture medium 12hours prior to the experiment and incubated for an additional 30 min at22° C., washed,solubilized in 10-20% SDS, and associated radioactivitydetermined in a scintillation β-counter. The number of attached cellswas quantitated by diving the cpm harvested by the cpm/cell. Under theseexperimental conditions, there was no aggregation of the variousleukocyte subpopulations in the presence of the different plasmaadhesive proteins, nor disruption of the HUVEC monolayers at any timeinterval tested, as judged by routine microscopic examination of theadhesion field. Data are the mean ± S.D. of triplicates from arepresentative experiment.

2) Time Dependency of Leukocyte-Endothelial Cell Interaction in thePresence of Vitronectin

Experimental conditions were the same as described in Example 9C1 exceptthat the ⁵¹Cr-labeled HL-60 cells were equilibrated with 40 μg/mlvitronectin or control protein transferrin and added to resting HUVECfor increasing time intervals between 10 and 60 minutes at 22° C. beforemeasurement of leukocyte adhesion.

3) Concentration Dependency of Leukocyte-Endothelial Cell Interaction inthe Presence of Vitronectin

Experimental conditions were the same as described in Example 9C1 exceptthat the ⁵¹Cr-labeled HL-60 cells were equilibrated with severalconcentrations of vitronectin ranging from 1.84 to 150 μg/ml and priorto incubation to resting HUVEC.

The results of these experiments are shown in FIG. 10. In FIG. 10A, thedata is plotted in a bar graph format with the numbers of attached⁵¹Cr-labeled HL-60 cells on the Y-axis. Terminally differentiated HL-60cells which had been stimulated with fMLP and equilibrated withfibrinogen, bound to HUVEC at a two to three-fold higher adhesion ratethan HL-60 cells which had not been equilibrated with an adhesiveprotein. These results are in agreement as previously described inLanguino et al., supra. Preincubation with comparable concentrations ofvitronectin enhanced the adhesion of differentiated HL-60 cells to HUVECthree to four-fold. Equilibration with fibronectin did not result in anincrease in adhesion. FIG. 10B and C demonstrate thatvitronectin-mediated increase in differentiated HL-60 cell adhesion toendothelium occurred in a time-and dose-dependent manner and wassaturated for physiologic plasma concentrations of vitronectin at 50μM/ml.

4) Binding Assays With THP-1 Cells

Binding assays were also performed as described in Example 10 C1described above with THP-1 cells. With comparable concentrations ofvitronectin, but not fibronectin, adhesion of monocytic THP-1 cells toresting HUVEC monolayers was demonstrated in a similar time- anddose-dependent manner.

The foregoing specification, including the specific embodiments andexamples, is intended to be illustrative of the present invention and isnot to be taken as limiting. Numerous other variations and modificationscan be effected without departing from the true spirit and scope of thepresent invention.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 5 <210> SEQ ID NO 1 <211> LENGTH: 411<212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE:<223> OTHER INFORMATION: expressed <220> FEATURE:<221> NAME/KEY: CARBOHYD <222> LOCATION: 88<223> OTHER INFORMATION: site of glycosylation <220> FEATURE:<221> NAME/KEY: DISULFID <222> LOCATION: (153)...(182)<223> OTHER INFORMATION: disulfide-bond <220> FEATURE:<221> NAME/KEY: DISULFID <222> LOCATION: (326)...(339)<223> OTHER INFORMATION: disulfide-bond <400> SEQUENCE: 1Tyr Val Ala Thr Arg Asp Asn Cys Cys Ile Le #u Asp Glu Arg Phe Gly 1               5   #                10   #                15Ser Tyr Cys Pro Thr Thr Cys Gly Ile Ala As #p Phe Leu Ser Thr Tyr            20       #            25       #            30Gln Thr Lys Val Asp Lys Asp Leu Gln Ser Le #u Glu Asp Ile Leu His        35           #        40           #        45Gln Val Glu Asn Lys Thr Ser Glu Val Lys Gl #n Leu Ile Lys Ala Ile    50               #    55               #    60Gln Leu Thr Tyr Asn Pro Asp Glu Ser Ser Ly #s Pro Asn Met Ile Asp65                   #70                   #75                   #80Ala Ala Thr Leu Lys Ser Arg Ile Met Leu Gl #u Glu Ile Met Lys Tyr                85   #                90   #                95Glu Ala Ser Ile Leu Thr His Asp Ser Ser Il #e Arg Tyr Leu Gln Glu            100       #           105       #           110Ile Tyr Asn Ser Asn Asn Gln Lys Ile Val As #n Leu Lys Glu Lys Val        115           #       120           #       125Ala Gln Leu Glu Ala Gln Cys Gln Glu Pro Cy #s Lys Asp Thr Val Gln    130               #   135               #   140Ile His Asp Ile Thr Gly Lys Asp Cys Gln As #p Ile Ala Asn Lys Gly145                 1 #50                 1 #55                 1 #60Ala Lys Gln Ser Gly Leu Tyr Phe Ile Lys Pr #o Leu Lys Ala Asn Gln                165   #               170   #               175Gln Phe Leu Val Tyr Cys Glu Ile Asp Gly Se #r Gly Asn Gly Trp Ile            180       #           185       #           190Val Phe Gln Lys Arg Leu Asp Gly Ser Val As #p Phe Lys Lys Asn Trp        195           #       200           #       205Ile Gln Tyr Lys Glu Gly Phe Gly His Leu Se #r Pro Thr Gly Thr Thr    210               #   215               #   220Glu Phe Trp Leu Gly Asn Glu Lys Ile His Le #u Ile Ser Thr Gln Ser225                 2 #30                 2 #35                 2 #40Ala Ile Pro Tyr Ala Leu Arg Val Glu Leu Gl #u Asp Trp Asn Gly Arg                245   #               250   #               255Thr Ser Thr Ala Asp Tyr Ala Met Phe Lys Va #l Gly Pro Glu Ala Asp            260       #           265       #           270Lys Tyr Arg Leu Thr Tyr Ala Tyr Phe Ala Gl #y Gly Asp Ala Gly Asp        275           #       280           #       285Ala Phe Asp Gly Phe Asp Phe Gly Asp Asp Pr #o Ser Asp Lys Phe Phe    290               #   295               #   300Thr Ser His Asn Gly Met Gln Phe Ser Thr Tr #p Asp Asn Asp Asn Asp305                 3 #10                 3 #15                 3 #20Lys Phe Glu Gly Asn Cys Ala Glu Gln Asp Gl #y Ser Gly Trp Trp Met                325   #               330   #               335Asn Lys Cys His Ala Gly His Leu Asn Gly Va #l Tyr Tyr Gln Gly Gly            340       #           345       #           350Thr Tyr Ser Lys Ala Ser Thr Pro Asn Gly Ty #r Asp Asn Gly Ile Ile        355           #       360           #       365Trp Ala Thr Trp Lys Thr Arg Trp Tyr Ser Me #t Lys Lys Thr Ile Met    370               #   375               #   380Lys Ile Ile Pro Phe Asn Arg Leu Thr Ile Gl #y Glu Gly Gln Gln His385                 3 #90                 3 #95                 4 #00His Leu Gly Gly Ala Lys Gln Ala Gly Asp Va #l                 405  #               410 <210> SEQ ID NO 2 <211> LENGTH: 17 <212> TYPE: PRT<213> ORGANISM: Unknown <220> FEATURE:<223> OTHER INFORMATION: synthesized <400> SEQUENCE: 2Asn Asn Gln Lys Ile Val Asn Leu Lys Glu Ly #s Val Ala Gln Leu Glu 1               5   #                10   #                15 Ala<210> SEQ ID NO 3 <211> LENGTH: 18 <212> TYPE: PRT<213> ORGANISM: Unknown <220> FEATURE:<223> OTHER INFORMATION: synthesized <400> SEQUENCE: 3Asn Asn Gln Lys Ile Val Asn Leu Lys Glu Ly #s Val Ala Gln Leu Glu 1               5   #                10   #                15 Ala Cys<210> SEQ ID NO 4 <211> LENGTH: 20 <212> TYPE: PRT<213> ORGANISM: Unknown <220> FEATURE:<223> OTHER INFORMATION: synthesized <400> SEQUENCE: 4Lys Tyr Gly Asn Asn Gln Lys Ile Val Asn Le #u Lys Glu Lys Val Ala 1               5   #                10   #                15Gln Leu Glu Ala             20 <210> SEQ ID NO 5 <211> LENGTH: 10<212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE:<223> OTHER INFORMATION: synthesized <400> SEQUENCE: 5Leu Gly Gly Ala Lys Gln Ala Gly Asp Val  1               5  #                10

What is claimed is:
 1. A method of inhibiting fibrinogen (Fg) binding toendothelial cells comprising contacting said endothelial cells with aFg-binding inhibiting amount of a physiologically acceptable compositioncomprising an antibody or an antigen binding fragment thereof thatimmunoreacts with amino acid residues 117-133 of fibrinogen γ chain,which amino acid sequence is shown in SEQ ID NO 2, wherein said antibodyor said antigen binding fragment thereof, is capable of binding tofibrinogen and inhibiting fibrinogen binding to endothelial cells.
 2. Amethod of claim 1 wherein said antibody is monoclonal.
 3. A method ofclaim 1 wherein said Fg-binding inhabiting amount is an amountsufficient to contact said endothelial cells with a concentration ofpoly peptide in the range of about 0.1 to 100 μg/ml.
 4. A method ofclaim 1 wherein said Fg-binding inhabiting amount is in the range ofabout 0.1 to about 20 milligrams of polypeptide per kilogram ofbodyweight of said patient per day.